Saccharomyces cerevisiae. Here, we report the development of Sinorhizobium meliloti, a nitrogen-fixing plant symbioticα-Proteobacterium, as a novel host that can store DNA, and mobilize DNA to E. coli, S. cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. To achieve this, we deleted the hsdR restriction-system in multiple reduced genome strains of S. meliloti that enable DNA transformation with up to 1.4 x 105 and 2.1 x 103 CFU μg-1 of DNA efficiency using electroporation and a newly developed polyethylene glycol transformation method, respectively. Multi-host and multi-functional shuttle vectors (MHS) were constructed and stably propagated in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum. We also developed protocols and demonstrated direct transfer of these MHS vectors via conjugation from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum. The development of S. meliloti as a new host for inter-kingdom DNA transfer will be invaluable for synthetic biology research and applications, including the installation and study of genes and biosynthetic pathways into organisms of interest in industry and agriculture.Storage, manipulation and delivery of DNA fragments is crucial for synthetic biology applications, subsequently allowing organisms of interest to be engineered with genes or pathways to produce desirable phenotypes such as disease or drought resistance in plants, or for synthesis of a specific chemical product. However, DNA with high G+C content can be unstable in many host organisms including  Saccharomyces cerevisiae [S. cerevisiae, such as bacterial conjugation. In addition, S. cerevisiae cannot maintain large DNA fragments with G+C content >40% without additional engineering [Streptomyces species have a G+C content >65% and are important for the production of antibiotics  [The field of synthetic biology aims to utilize existing or novel biological parts and systems to create organisms that can help address global problems including increased demand for food, fuel, therapeutics, and high value chemicals. However, one of the major obstacles in synthetic biology is that many organisms of interest lack genetic tools such as autonomously replicating plasmids, well characterized promoters and terminators, selective markers, genome-editing tools, and protocols to uptake and install DNA ,2. This revisiae –10, howeineering –13, and e, etc.) . TherefoSinorhizobium meliloti is an attractive host candidate for this application. S. meliloti is a Gram-negative α-Proteobacterium that forms a symbiotic relationship with legume plants where it fixes nitrogen in root nodules, and therefore is highly important in agriculture. S. meliloti model strain Rm1021 has a multipartite genome including a chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb) [S. meliloti now exist that vary in genome size, nutrient requirements and generation time [S. meliloti with a minimal genome lacking the pSymA and pSymB replicons was developed, resulting in a 45% reduction of the genome [engA and tRNAarg, and these genes were transferred to the main chromosome [repABC origins taken from various α-Proteobacteria that can be propagated in S. meliloti [S. meliloti at one time, along with the endogenous pSymA and pSymB replicons. Utilizing these vectors, an in vivo cloning method with Cre/lox-mediated translocation of large DNA fragments to the repABC-based vector was established [S. meliloti is an attractive host organism due to its high G+C content genome (62%) [1.68 Mb) . Multiplion time . Recentle genome . Two essromosome . The repromosome , and canmeliloti . Three oablished . Therefome (62%) , availabS. meliloti is also an attractive host bacterium because it is able to move DNA to other organisms using several methods. Previous studies have demonstrated DNA transfer methods from S. meliloti through introduction of a Ti plasmid to plant species through mobilization of TDNA [S. meliloti is able to move DNA to bacteria via conjugation in tri-parental mating experiments [ of TDNA . In addieriments .S. meliloti, such as pTA-Mob, will allow for faster and more efficient transfer of DNA to a recipient organism. The conjugative pTA-Mob plasmid, which contains all of the genes required for direct DNA transfer via conjugation, can mobilize any additional cargo plasmid containing an origin of transfer (oriT). Conjugation is a particularly attractive method of DNA transfer to streamline the workflow for genetic manipulation of organisms. While transfer of DNA from S. meliloti to bacterial cells has been demonstrated, DNA transfer to eukaryotic cells, such as yeast and algae, has not been previously described.Installing a conjugative plasmid into S. meliloti [S. meliloti pSymA and pSymB replicons to create multi-host shuttle (MHS) vectors that replicate in S. meliloti, Escherichia coli, S. cerevisiae, and Phaeodactylum tricornutum. The MHS vectors were tested for their ability to provide antibiotic resistance and propagate in S. meliloti. These vectors were moved into S. meliloti strains via an optimized electroporation protocol, via a newly developed polyethylene glycol (PEG) method, or via conjugation from E. coli. Most notably, we have developed protocols to directly transfer the MHS vectors via conjugation from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum.Here, we utilized the reduced genome strains of meliloti  to creatmeliloti  plasmid S. meliloti strains to remove the restriction-system (ΔhsdR) to allow for development of more efficient transformation methods. Disruption or deletion of the hsdR gene has been previously reported in the Rm1021 strain of S. meliloti, and deletion mutants were shown to have enhanced transformation efficiencies [S. meliloti strains, either lacking one or both of the pSymA or pSymB replicons, the hsdR gene was replaced by a FRT-Km/Nm-FRT cassette and the resulting ΔhsdR::Nm mutant allele was transferred to various background strains via transduction of Nmr. The FRT-Km/Nm-FRT cassette was then removed by introducing an unstable plasmid (pTH2505) carrying the Flp recombinase, followed by curing of this plasmid. The hsdR deletion strains were verified to have the hsdR gene successfully deleted using diagnostic PCR with primers located upstream and downstream of the hsdR gene locus  [E. coli after conjugation to S. meliloti or any other organism.Additionally, a designer id (DAP) , therefoS. meliloti created, and the origins of replications of the large megaplasmid and chromid (repA1B1C1 and repA2B2C2) identified, MHS vectors were developed as cargo plasmids for conjugation from S. meliloti to various recipients. Six MHS vectors were designed and constructed to allow for stable replication and selection in our S. meliloti conjugative donor, as well as several conjugative recipient organisms including E. coli, S. cerevisiae, and P. tricornutum. These organisms were chosen as conjugative recipients as they are well-characterized model strains for bacterial, yeast and algal systems. These vectors were constructed with a bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) backbone allowing for replication in E. coli and S. cerevisiae [P. tricornutum [S. meliloti, pSymA , or the pSymB chromid . Selectable markers include spectinomycin (Sp), tetracycline (Tc), or kanamycin/neomycin (Km/Nm) resistance for S. meliloti , nourseothricin N-acetyl transferase (Ntc) for P. tricornutum [S. cerevisiae, and chloramphenicol (Cm) resistance for E. coli. Finally, all MHS vectors contain an origin of transfer (oriT) that is acted on by proteins encoded on the conjugative plasmid pTA-Mob, and is necessary for mobilization of the MHS vectors from S. meliloti to recipient organisms , pAGE2.0 (Tc), and pAGE3.0 (Km/Nm), were constructed with the pSymA origin (repA2B2C2). An additional three pBGE vectors differing only in the S. meliloti selective marker, pBGE1.0 (Sp), pBGE2.0 (Tc), and pBGE3.0 (Km/Nm), were constructed with the pSymB origin (repA1B1C1). Once assembled [E. coli Epi300 (Epicentre) cells, isolated and confirmed to be correctly assembled by a diagnostic restriction digest  after restreaking from non-selective media (LBmc 38 μM FeCl3 2 μM CoCl2)  produced efficiencies averaging 1.4 x 105 CFU μg-1 of DNA  from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum (repA2B2C2) and the spectinomycin selectable marker for S. meliloti. Conjugation efficiencies were calculated as the number of transconjugants per recipient cell from the S. meliloti conjugative donor to E. coli, S. cerevisiae, and P. tricornutum as 2.22 x 10−1, 7.99 x 10−6 and 9.40 x 10−5, respectively  and 30 E. coli transformants (each transformed with DNA from independent P. tricornutum colonies) were selected, induced with 0.1% arabinose, and the vector DNA was isolated. Diagnostic restriction digests were performed on these vectors using EcoRV-HF, and the number of vectors with the expected banding pattern was 18/20 originating from E. coli, 19/20 originating from S. cerevisiae and 16/30 originating from P. tricornutum  supplemented with appropriate antibiotics (streptomycin (100 μg mL-1), spectinomycin (200 μg mL-1), gentamicin (40 μg mL-1), tetracycline (5 μg mL-1), neomycin (100 μg mL-1)), and 38 μM FeCl3 and/or 2 μM CoCl2, when appropriate. Saccharomyces cerevisiae VL6−48 (ATCC MYA-3666: MATα his3-Δ200 trp1-Δ1 ura3−52 lys2 ade2−1 met14 cir0) was grown at 30°C in rich medium (2X YPD) supplemented with 80 mg L-1 adenine hemisulfate, or yeast synthetic complete medium lacking histidine supplemented with 60 mg L-1 adenine sulfate  [Escherichia coli  was grown at 37°C in Luria Broth (LB) supplemented with appropriate antibiotics (chloramphenicol (15 μg mL-1)). Escherichia coli (ECGE101) was grown at 37°C in Luria Broth (LB) supplemented with appropriate antibiotics (chloramphenicol (15 μg mL-1), gentamicin (40 μg mL-1)), and diaminopimelic acid (60 μg mL-1). Phaeodactylum tricornutum (Culture Collection of Algae and Protozoa CCAP 1055/1) was grown in L1 medium without silica at 18°C under cool white fluorescent lights (75 μE m-2 s-1) and a photoperiod of 16 h light:8 h dark [a, Inc.) . EscheriS. meliloti strains used in this study were created by taking the strain RmP3500, which lacks pSymA and pSymB where the engA-tRNAArg-rmlC genomic region has been introduced into the chromosome [hsdR::Nm, NmR SmR.Designer romosome  and re-ihsdR restriction gene has been replaced by a Nm resistance cassette. This strain was created by first PCR amplifying a downstream region and upstream region of the hsdR gene and cloning these PCR products into the XbaI site of pUCP30T using SLiC [R) with the hsdR upstream/downstream regions was then transformed into an E. coli strain harboring pKD46 (AmpR) which includes genes for lambda red recombinase, the resulting strain was named M2453. A Km/Nm cassette for hsdR deletion was then PCR amplified using high-fidelity DNA polymerase and purified. The cassette consisted of the antibiotic resistance gene flanked by regions of homology to the hsdR regions cloned into pUCP30T. The cassette was then electroporated into competent M2453 cells (grown in 1mM Arabinose to induce lambda red recombinase genes) and selected using Km (25 μg mL-1). Cells were then patched on Km (25 μg mL-1), Gm (10 μg mL-1) and Amp (100 μg mL-1). A KmR, GmR, and AmpS strain was then streak purified and named M2459. The resulting vector was then conjugated into RmP110 as recipient and M2459 as donor. Selection was done in Sm (200 μg mL-1) Nm (200 μg mL-1) plates. Resulting transconjugants were then patched on Gm (10 μg mL-1), (Nm 200 μg mL-1) and Sm (200 μg mL-1) plates and a Gm sensitive colony was streak purified (indicating double recombination of cassette with hsdR locus). The strain was then verified using diagnostic primers that spanned the hsdR upstream/downstream region with the Km/Nm cassette.RmP3975 is a strain where the ing SLiC , then trS. meliloti strains containing various combinations of pSymA and pSymB and selecting for Nm resistance. The Nm resistance cassette was then excised by introducing Flp recombinase to flip out the cassette using flanking FRT sites. The resulting strains are are RmP4258 (pSymA- pSymB- ΔhsdR), RmP4260 (pSymA+ pSymB- ΔhsdR), RmP4124 (pSymA- pSymB+ ΔhsdR), and RmP4125 (pSymA+ pSymB+ ΔhsdR).The Nm resistance cassette was then able to be transduced into the dapA gene replacement with lambda pir and an erythromycin cassette from strain βDH10B was replicated in Epi300, resulting in a strain containing trfA and requirement of diaminopimelic acid (DAP) supplementation for growth. This is useful because it allows for replication of plasmids with RK2 oriV and makes it a convenient donor in biparental conjugation because the DAP requirement eliminates the need for antibiotic counter-selection. A lambda red recombinase plasmid (pKD46) was electroporated into Epi300, because Epi300 is recA-. The dapA region from βDH10B was amplified using the flanking primers DAP1 and DAP2. The fragment was electroporated into E. coli Epi300 containing pKD46 and transformants were selected on LB with DAP (60 μg mL-1) and erythromycin (200 μg mL-1), and inability to grow in the absence of DAP was confirmed. Such transformants were cured of pKD46 by growing at 37°C and confirming ampicillin (100 μg mL-1) sensitivity.The E. coli. Additionally, it is low copy vector that can be induced to high copy with arabinose. Other components amplified for vector assembly include the antibiotic resistance cassettes for selection in S. meliloti, repA2B2C2 or repA1B1C1 for replication in S. meliloti, origin of transfer (oriT) for conjugation, and selective marker for algae (Ntc). The six fragments were PCR amplified and assembled in yeast using a yeast spheroplast transformation method. Next, DNA was isolated from yeast and moved to E. coli strain PG5alpha and genotyped using a multiplex PCR screen. Correct vectors were moved from PG5alpha to Epi300 and Epi300 pTA-Mob. Diagnostic digests were performed to confirm correct assembly of vectors.Briefly, pAGE and pBGE vectors were constructed based on the pCC1BAC vector to which elements for replication in yeast were added. This backbone was amplified from plasmid p0521s (created at Venter institute and available on Addgene). This will allow replication and selection in yeast and hsdR strains to maintain these large pAGE vectors, stability assays were performed to determine how long pAGE2.0 can be maintained in RmP4122 ∆pSymA∆hsdRNms. This was performed in triplicate. Single colonies harboring pAGE2.0 were inoculated into LBmc FeCl3 + CoCl2 with Tc (5μg mL-1) at 30°C. The next day, 100uL of culture diluted to 10−6 was plated on LBmc FeCl3 + CoCl2 and grown 3 days at 30°C. Cultures were subcultured 1000X in LBmc FeCl3 + CoCl2 without antibiotics and grown overnight. Approximately 10 doublings occurred per day. The next day, cultures were diluted to 10−6 and again 100μL was plated on LBmc FeCl3 + CoCl2. Cultures were subcultured again as before. When visible, 100 colonies were patched onto LBmc FeCl3 + CoCl2 with Tc (5μg mL-1) and without Tc as a control to ensure colony viability. These plates were incubated at 30°C for 3 days. The number of patched colonies that were able to grow on selective media was then recorded. The experiment was performed for 5 days to assess stability of pAGE2.0.To characterize the ability of the ΔS. meliloti overnight in LBmc, 38 μM FeCl3, and streptomycin 100 μg mL-1 at 30°C with shaking incubation to an OD600 of 2.0. Incubate flask on ice for 10 min then pellet at 6000 x g at 4°C for 10 min. Resuspend cells in 250 mL of sddH2O by gentile agitated in a water bath, top up volume to 500 mL, then pellet with the same conditions. Repeat this wash step with sddH2O two additional times, and then repeat once more with the same volume of 10% glycerol. Resuspend the pellet in 3 mL of 10% glycerol, flash freeze 200 μL aliquots and store at -80°C.Grow 500 mL of 3 and recover in a test tube at 30°C with shaking incubation (225rpm) for 120 min. Spread 500 μL of the transformation mixture on LBmc plates containing 38 μM FeCl3, 100 μg mL-1 streptomycin, and an appropriate concentration of antibiotic selection based on the transformed DNA. Incubate plates at 30°C for 3 days to allow for colony formation.Incubate frozen cells on ice until fully thawed (about 15 min). Add 50 ng (in 1 μL) of DNA to 50 μL of competent cells in a 1.5 mL Eppendorf tube on ice, flick to mix, and incubate on ice for 5 min. Add transformation mixture to a 0.1 mm path length cuvette on ice and electroporate at 1.8 kV. Immediately add 1 mL LBmc 38μM FeClS. meliloti cells were cultured overnight in LBmc, 38μM FeCl3, and 100 μg mL-1 streptomycin at 30°C with shaking incubation (225rpm). Upon reaching OD600 = 0.4, cells were harvested into sterile 500 mL centrifuge bottles, incubated on ice for 10 minutes, and pelleted at 4000 x g and 4°C for 10 minutes. Cells were then resuspended in 100 mL of ice-cold 100 mM CaCl2 by gentle pipetting and incubated on ice for an additional 30 minutes. Following incubation, cells were pelleted again at 4000 x g and 4°C for 10 minutes and resuspended in 1.25 mL of ice-cold 100 mM CaCl2 + 15% glycerol. The final resuspension volume was split into 25 μL aliquots, flash frozen using liquid nitrogen, and stored at -80°C for later use.3 was added to the reaction tube and cells were recovered at 30°C with shaking incubation (225rpm) for 90 minutes. Following recovery, 250 μL of the transformation mixture were spread on LBmc plates containing 38 μM FeCl3, 100 μg mL-1 streptomycin, and an appropriate concentration of antibiotic selection. Plates were incubated at 30°C for 3 days to allow for colony formation.Frozen cells  were incubated on ice until fully thawed. Next, 200 ng (in 3 μL) of supercoiled pAGE DNA and 25 μL of 10% PEG 4000 were then added to the reaction tube and mixed evenly by gentle pipetting. Cells were then incubated on ice for 30 minutes, transferred to a 40°C water bath for 8 minutes, and immediately placed back on ice for 10 minutes. Following, 500 μL of LBmc, 38μM FeCl600 = 2.0) in LBmc, 38μM FeCl3, streptomycin 100 μg mL-1 was diluted 20x to make 20 mL culture and grown 6 hours shaking at 30°C in LBmc supplemented with streptomycin 100 μg mL-1 and Fe to OD600 of 0.9. The culture was diluted 2000X and grown with shaking at 30°C in 50 mL LBmc supplemented with streptomycin 100 μg mL-1 and Fe to OD600 of 0.6. The culture was centrifuged for 10 min at 6,000 x g at 4°C and resuspended in 300 μL of LBmc media.An overnight culture  was plated on LBmc plates supplemented with Fe, streptomycin 100 μg mL-1, and spectinomycin 200 μg mL-1 (Note: plates should be at least 35 mL thick). Plates were incubated at 30°C for 3 days before colonies were counted.First, 50 μL of 600 = 2.0) was diluted 20X to make 20 mL culture and grown 6 hours shaking at 30°C in LBmc supplemented with streptomycin 100 μg mL-1 spectinomycin 200 μg mL-1, gentamicin 40 μg mL-1 and Fe to OD600 of 0.3. The culture was diluted 500X and grown with shaking at 30°C in 50 mL LBmc supplemented with streptomycin 100 μg mL-1, spectinomycin 200 μg mL-1, gentamicin 40 μg mL-1 to OD600 of 0.6. The culture was centrifuged for 10 min at 6,000 x g at 4°C and resuspended in 300 μL of LBmc media.Stock culture  was plated on LB plates supplemented with chloramphenicol 15 μg mL-1. Plates were incubated at 37°C for 16 hours before colonies are counted.First, 50 μL of 600 = 2.0) was diluted 20x to make 20 mL culture and grown 6 hours shaking at 30°C in LBmc supplemented with streptomycin 100 μg mL-1, spectinomycin 200 μg mL-1, gentamicin 40 μg mL-1 and 38μM FeCl3 to OD600 of 0.3. The culture was diluted and grown with shaking at 30°C in 120 mL LBmc supplemented with streptomycin 100 μg mL-1, spectinomycin 200 μg mL-1, gentamicin 40 μg mL-1, acetosyringone 100 μg mL-1 to OD600 of 2.0 . The culture was centrifuged for 10 min at 6,000 x g at 4°C and resuspended in 1.5 mL of LBmc media.Stock culture . Then plates were incubated for 180 minutes at 30°C. Then, 2 mL of sddH20 was added to plates and cells were scraped. Next, 100 μL of the scraped cells was plated on 2% -HIS plates supplemented with ampicillin 100 μg mL-1. Plates were incubated at 30°C where colonies start to appear after 2–3 and colonies are counted after 5 days.First, 200 μL of Note: For simplicity, accetosyringone steps can be removed from the final protocol without any substantial decrease in conjugation efficiency.8 cells mL-1 using counts from a hemocytometer, was plated on ½L1 1% agar plates and grown for 4 days. Then, 1 mL of L1 media was added to the plate and cells were scraped, counted using a hemocytometer, and adjusted to a concentration of 1 x 109 cells mL-1.First, 250 μL of liquid grown culture was adjusted to 1.0 x 10600 = 2.0) was diluted 20x to make 20 mL culture and grown 6 hours shaking at 30°C in LBmc supplemented with spectinomycin 200 μg mL-1, gentamicin 40 μg mL-1 and Fe to OD600 of 0.3. The culture was diluted 25X and grown for 12 hours with shaking at 30°C in 50 mL LBmc supplemented with spectinomycin 200 μg mL-1, gentamicin 40 μg mL-1 to OD600 of 0.6. The culture was centrifuged for 10 min at 5,000 x g at 4°C and resuspended in 500 μL of LBmc media.Stock culture (ODP. tricornutum cells and 200 μL of S. meliloti cells were mixed directly on ½L1 10% LBmc 1% agar plates (Note: plates are dried in the biosafety cabinet for one hour before conjugation) and incubated for 180 minutes at 30°C in the dark, then moved to 18°C in the light and grown for 2 days. After two days, 2 mL of L1 media was added to plates, cells were scraped, and 100 μL (5%) was plated on ¼L1 1% agar plates supplemented with nourseothricin 100 μg mL-1, and ampicillin 100 μg mL-1 (Note: plates should be at least 35 mL thick). Plates were incubated at 18°C in the light/dark cycle and colonies start to appear after 7 days and are allowed to develop to 14 days before colonies are counted.First, 200 μL of 9 cells mL-1.First, 200 μL of liquid grown culture was diluted using counts from a hemocytometer, and grown in ½L1 media for 4 days. Cell counts from a hemocytometer were used and culture was pelleted at 4000 x g 10 min 4°C, and adjusted to a concentration of 1 x 10600 = 2.0) was diluted 20x to make 20 mL culture and grown 6 hours shaking at 30°C in LBmc supplemented with spectinomycin 200 μg mL-1, gentamicin 40 μg mL-1 and Fe to OD600 of 0.3. The culture was diluted 25X and grown for 12 hours with shaking at 30°C in 50 mL LBmc supplemented with spectinomycin 200 μg mL-1, gentamicin 40 μg mL-1 to OD600 of 0.6. The culture was centrifuged for 10 min at 5,000 x g at 4°C and resuspended in 500 μL of LBmc media.Stock culture (ODP. tricornutum cells and 5 μL of S. meliloti cells were mixed together in a 96-well plate. The mixture (10 μL) was transferred to a 96-well plate containing 200 μL of ½L1 10% LBmc 1% agar (note: plates are dried in the biosafety cabinet for one hour before conjugation). This conjugation plate was incubated for 180 minutes at 30°C in the dark, then moved to 18°C in the light and grown for 2 days. After two days, 100 μL of L1 media was added to wells and cells were scraped (X2), and 10 μL (5%) was plated on ¼L1 1% agar supplemented with nourseothricin 100 μg mL-1, and ampicillin 100 μg mL-1 in a 96-well plate. Plates were incubated at 25°C in the light/dark cycle for 24 hours and then 18°C in the light/dark cycle for an additional 24 hours. Colonies start to appear after 7 days and are allowed to develop up to 14 days before colonies are counted.First, 5 μL of E. coli using the BioBasic EZ-10 miniprep kit. Plasmid DNA was isolated from all other species using a modified alkaline lysis protocol. Plasmid DNA was isolated from all other species and E. coli containing plasmids >60 kb using the modified alkaline lysis protocol described below. Steps 1–3 are variable depending on the species, while steps 4–10 are common for all species. Steps 1–3 for E. coli and S. meliloti. (1) Five mL cultures were grown to saturation overnight. (2) Cells were pelleted at 5000 x g for 10 min at 4°C, and the supernatant was discarded. (3) Cells were resuspended in 250 uL of resuspension buffer (which contained 240 ml P1 (Qiagen), 5 ml of 1.4 M b-Mercaptoethanol and 5 ml Zymolyase solution (Zymolyase solution: 200 mg Zymolyase 20 T (USB), 9 ml H2O, 1 ml 1 M Tris pH7.5, 10 ml 50% glycerol, stored at 20°C). Steps 1–3 for S. cerevisiae. (1) Five mL of culture was grown to saturation. (2) Cells were pelleted at 5000 x g for 10 min 10 min at 4°C, and the supernatant was discarded. (3) Cells were resuspended in 250 mL resuspension buffer (as described above) and incubated at 37°C for 60 min. Steps 1–3 for P. tricornutum. (1) Five mL cultures were harvested during exponential growth phase. (2) Cells were pelleted at 4,000g for 10 min at 4°C, and the supernatant was discarded. (3) Cells were resuspended in 250 mL resuspension buffer, which contained 235 ml P1 (Qiagen), 5 ml hemicellulase 100 mg ml 1, 5 ml of lysozyme 25 mg ml 1, and 5 ml Zymolyase solution (Zymolyase solution: 200 mg Zymolyase 20T (USB), 9 ml H2O, 1 ml 1 M Tris pH7.5, 10 ml 50% glycerol, stored at 20°C) and then cells were incubated at 37°C for 30 min. Steps 4–10 common for all species. (4) 250 uL of lysis buffer P2 (Qiagen) was added and samples were inverted 5–10 times to mix. (5) 250 ml of neutralization buffer P3 was added and samples were inverted 5–10 times to mix. (6) Then samples were spun down at 16,000g, 10 min at 4°C (7) Supernatant was transferred to a clean tube and 750 ul ice-cold isopropanol was added and the samples were mixed by inversion and spun down at 16,000g, 10 min at 4°C (8) Next the supernatant was removed and 750 ul ice-cold 70% EtOH was added and samples were mixed by inversion and spun down at 16,000g, 5 min. (9) Next the supernatant was discarded, pellets were briefly dried and resuspended in 50 uL of TE buffer. (10) After that the samples were kept at 37°C for 30–60 min to dissolve.Plasmid DNA (<60 kb) was isolated from S1 FilehsdR deletion in reduced S. meliloti strains by diagnostic PCR. Figure B. Diagnostic restriction digest of pAGE and pBGE vectors. Figure C. Vector stability assay of pAGE2.0 in S. meliloti over 50 generations. Figure D. Workflow of optimized electroporation transformation protocol for S. meliloti. Figure E. Workflow of optimized PEG-mediated transformation protocol for S. meliloti. Figure F. PEG-mediated transformation of pAGE vectors into S. meliloti. Figure G. EcoRV-HF diagnostic digest of pAGE1.0 vectors extracted from 20 E. coli colonies following conjugation from S. meliloti to E. coli. Figure H. EcoRV-HF diagnostic digest of pAGE1.0 vectors extracted from 20 E. coli colonies following conjugation from S. meliloti to P. tricornutum. Figure I. EcoRV-HF diagnostic digest of pAGE1.0 vectors extracted from 20 E. coli colonies following conjugation from S. meliloti to S. cerevisiae.Figure A. Verification of (DOCX)Click here for additional data file.S2 FileS. meliloti RmP4122 ∆pSymA ΔhsdR. Table B. Efficiency of DNA transfer methods to S. meliloti RmP4122 ∆pSymA ΔhsdR including PEG-mediated transformation, electroporation and conjugation of pAGE vectors. Table C. Efficiency of pAGE1.0 conjugation from S. meliloti to recipient organisms and ratio of donor to recipient cells involved. Table D. Analysis of pAGE1.0 vectors recovered from conjugations from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum. Table E. List of oligonucleotides used in this study. Table F. List of strains used in this study. Table G. List of vectors used in this study.Table A. Vector stability of pAGE2.0 in (DOCX)Click here for additional data file.


Escherichia coli host. However, the functions of many of these proteins are not known. In this study, we have characterized the T4 early gene motB, located in a dispensable region of the T4 genome. We show that heterologous production of MotB is highly toxic to E. coli, resulting in cell death or growth arrest depending on the strain and that the presence of motB increases T4 burst size 2-fold. Previous work suggested that motB affects middle gene expression, but our transcriptome analyses of T4 ammotB vs. T4 wt infections reveal that only a few late genes are mildly impaired at 5 min post-infection, and expression of early and middle genes is unaffected. We find that MotB is a DNA-binding protein that binds both unmodified host and T4 modified  DNA with no detectable sequence specificity. Interestingly, MotB copurifies with the host histone-like proteins, H-NS and StpA, either directly or through cobinding to DNA. We show that H-NS also binds modified T4 DNA and speculate that MotB may alter how H-NS interacts with T4 DNA, host DNA, or both, thereby improving the growth of the phage.The lytic bacteriophage T4 employs multiple phage-encoded early proteins to takeover the  An increase in the occurrence of antibiotic-resistant bacteria has sparked an interest in phage–host interactions ,2. As baEscherichia coli, resulting in rapid cell lysis after ~20 min at 37 °C. Despite the use of T4 as a model organism for decades, the functions of many genes, especially those expressed early during infection, remain unknown [55 (gene product [gp]55), recognizes this element, facilitating late transcription along with the coactivator, gp33, and the sliding clamp, gp45 (reviewed in ATP. 32P-labeled single stranded (ss) DNA, purified by phenol extraction/butanol precipitation, was resuspended in 1× TE . Double-stranded (ds) oligonucleotides were prepared by mixing the 32P-labeled top strand with the complementary bottom strand in 1× OptiKinase Reaction Buffer , heating at 92 °C for 2 min, and slowly cooling to room temperature. The 32P-labeled dsDNA was subsequently purified using a G-25 microspin column .Oligonucleotides used for primer extensions, RT-qPCR, and gel retardation assays were synthesized by Integrated DNA Technologies ; sequences are available upon request. Radiolabeled oligonucleotides were prepared by treating the top strand oligonucleotide with OptiKinase  in the presence of ATP prior to PCR. The PCR product was isolated after gel electrophoresis using an Elutrap  and ethanol precipitated.The E. coli strains TOP10F’ , BL21(DE3), and BL21(DE3)/pLysE [E. coli were used for T4 infections. Unless otherwise noted, cells were grown at 37 °C with shaking at 250 rpm.3)/pLysE  were use+ (T4 wt), T4 amG1 (T4 ammotA) [ammotB were used for infections. T4 ammotB was obtained by recombination of pTE103-ammotB into the T4 genome. BL21(DE3)/pTE103-ammotB was infected with T4 wt at a multiplicity of infection (MOI) of 1 during exponential phase and incubated for 75 min. Cells were lysed with chloroform and resulting phage were titered on NapIV S. Plaques were screened for the presence of the amber mutation by hybridization of a 32P-labeled probe containing either the wt or mutant sequence as described [ammotB. To confirm the presence of the amber mutation, the motB gene was amplified by PCR then sequenced by Macrogen .Wild-type T4D motAam) , and T4 escribed . Potentiw/v) LB agar  containing 40 µg/mL kanamycin, 12 µg/mL tetracycline (TOP10F’ only), and 0.5% (w/v) glucose. Overnight cultures were grown in LB  containing 40 µg/mL kanamycin, 12 µg/mL tetracycline (TOP10F’ only), and 0.025% (w/v) glucose. Overnight cultures were diluted to an OD600 of 0.1 with LB containing 40 µg/mL kanamycin and 12 µg/mL tetracycline (TOP10F’ only). At an OD600 of approximately 0.3, 0.2% (w/v) arabinose  was added to each sample. At the indicated times samples were taken and electrophoresed on SDS-PAGE gels to monitor MotB production.BL21(DE3) and TOP10F’ containing pNW129 (empty vector) or pNW129-MotB were plated on 1.5%  for 2 h. Cells were harvested by centrifugation at 13,000× g for 10 min and stored at −80 °C. Unless otherwise noted, the following procedures were performed on ice or at 4 °C. Cells were resuspended in CB Buffer (20 mM HEPES-OH (pH 8.5), 50 mM NaCl, 1 mM EDTA, 0.01% (v/v) Triton X-100) containing 1 mM benzamidine, then lysed by sonication until OD600 was reduced ~3-fold. Clarified supernatant was obtained by centrifugation at 15,000× g for 30 min followed by filtration through a 0.4 µm syringe filter. Chitin resin (New England BioLabs) equilibrated with CB Buffer (5 mL slurry per 125 mL starting culture) was added to the supernatant, and the suspension was gently rocked overnight. Resin was transferred to a 30 mL disposable column  and then washed with ~25 column volumes of CB Buffer containing 1 M NaCl at a flow rate of <2 mL/min (gravity drip) until the A260 reached a minimum. The resin was subsequently washed with an additional 7 column volumes of CB Buffer containing 500 mM NaCl. On-column cleavage of the Intein/CBD-tag was initiated by quickly washing the column with 3 column volumes of CB Buffer containing 500 mM NaCl and 1 mM dithiothreitol (DTT). After ≥40 h, MotB was eluted by the addition of 2 column volumes of CB Buffer containing 500 mM NaCl and 2 mL fractions were collected. Fractions containing MotB were identified by SDS-PAGE and then dialyzed into Buffer A (25 mM Tris-HCl (pH 8.0), 50 mM NaCl, 6 M deionized Urea). The denatured MotB was isolated after passage through a 1 mL HiTrap Q column , in which MotB was in the flow-through fraction while residual nucleic acid bound to the column. MotB was then refolded using the dialysis procedure previously described for E. coli σ70 renaturation [v/v Triton X-100, 50% v/v glycerol, 0.1 mM DTT).MotB containing a C-terminal intein tag with a chitin binding domain (MotB-Intein/CBD) was isolated and purified from BL21(DE3)/pLysE containing pTXB1-MotB. Overnight cultures were grown in LB containing 25 µg/mL chloramphenicol and 100 µg/mL carbenicillin. Cells were diluted to an ODturation  and stor6-tag (MotB-His) was isolated and purified from TOP10F’ containing pNW129-MotB-His. Overnight cultures were grown in LB containing 12 µg/mL tetracycline, 40 µg/mL kanamycin, and 0.05% (w/v) glucose. Cells were diluted to an OD600 ~0.1 in LB containing 12 µg/mL tetracycline and 40 µg/mL kanamycin and grown to an OD600 around 0.3, when synthesis of MotB-His was induced by the addition of 0.2% (w/v) arabinose  for 1 h. Cells were harvested by centrifugation at 13,000× g for 10 min and stored at −80 °C. Unless otherwise noted, the following procedures were performed on ice or at 4 °C. Cells were resuspended in 40 mL of Lysis Buffer (25 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM imidazole, 0.01% (v/v) Triton X-100, 1 mM benzamidine) per 1 L culture, lysed by sonication as described above and then centrifuged at 17,500× g for 20 min. The pellet, containing MotB-His, was resuspended in Lysis Buffer and centrifuged again at 17,500× g for 15 min. To recover MotB-His, the pellet was resuspended in 20 mL Binding Buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole, 6 M urea, 1 mM benzamidine, pH adjusted to 7.9) per 1 L culture, incubated for 1 h with gentle mixing, and then centrifuged at 17,500× g for 15 min. The supernatant, containing MotB-His, was then incubated with gentle mixing for a minimum of 1 h with 5 mL (per 1 L culture) Ni-NTA His-binding resin  that was pre-equilibrated with Binding Buffer. Resin was transferred to a 30 mL disposable column (Bio-Rad) then washed with four column volumes of Binding Buffer followed by six column volumes of Wash Buffer (25 mM Tris-HCl (pH 8.0), 500 mM NaCl, 60 mM imidazole, 6 M urea, pH adjusted to 7.9) at a flow rate of <2 mL/min (gravity drip). The major fraction of MotB-His eluted with 2 column volumes of Elution Buffer 1 (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 200 mM imidazole, 6 M urea, pH adjusted to 7.9). Residual MotB-His was subsequently eluted with 2 column volumes of Elution Buffer 2 (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 400 mM imidazole, 6 M urea, pH adjusted to 7.9). For both elution conditions, 2.5 mL fractions were collected. Fractions containing MotB-His, identified by SDS-PAGE, were refolded as described in Method I.MotB containing a C-terminal His600 ~0.1 in LB containing 25 µg/mL chloramphenicol and 100 µg/mL carbenicillin and then grown at 25 °C with shaking at 250 rpm. At an OD600 between 0.3 and 0.4, synthesis of H-NS-Intein/CBD was induced by the addition of 4 mM IPTG  for 2 h. Cells were harvested by centrifugation at 13,000× g for 10 min and stored at −80 °C. Unless otherwise noted, the following procedures were performed on ice or at 4 °C. Cells were resuspended in 30 mL of H-NS Sonication Buffer (20 mM Tris-HCl (pH 8.0), 750 mM NaCl, 1 mM EDTA, 0.01% (v/v) Triton X-100, 1 mM Benzamidine) then lysed by sonication until OD600 was reduced ~3-fold. Clarified supernatant was obtained by centrifugation at 15,000× g for 30 min followed by filtration through a 0.45 μm syringe filter. Chitin resin (New England BioLabs) equilibrated with H-NS Sonication Buffer (5 mL slurry per 250 mL starting culture) was added to the supernatant and rocked overnight. Resin/cell lysate was transferred to a 30 mL disposable column (Bio-Rad) and washed with approximately 20 column volumes of CCB buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 0.01% (v/v) Triton X-100) at a flow rate of <2 mL/min (gravity drip). On-column cleavage of the Intein/CBD-tag was initiated by quickly washing the column with 3 column volumes of CCB buffer containing 50 mM DTT. After ≥40 h, H-NS was eluted by the addition of 3 column volumes of CCB buffer and 1 mL fractions were collected. Fractions containing H-NS were identified by SDS-PAGE and pooled. As this pool contained a small amount of intein tagged protein, it was passed over a new column containing 5 mL of fresh, equilibrated chitin slurry, and fractions from the flow-through plus 2 column volume washes were collected. The now pure H-NS was dialyzed into H-NS Storage Buffer (10 mM potassium phosphate (pH 7.5), 200 mM NaCl, 0.1 mM EDTA, 50% (v/v) glycerol) and stored at −20 °C.H-NS containing a C-terminal intein tag with a chitin binding domain (H-NS-Intein/CBD) was isolated and purified from BL21(DE3)/pLysE containing pTXB1-H-NS. Overnight cultures were grown in LB containing 25 µg/mL chloramphenicol and 100 µg/mL carbenicillin. Cells were diluted to an ODMotB samples used to identify copurifying nucleic acid were purified using MotB purification Method I with the following modifications. Elution fractions from the chitin column that contained MotB as determined by SDS-PAGE were directly dialyzed into MotB Storage Buffer and stored at −80 °C. Protein was removed by chloroform:isoamyl alcohol (24:1) extraction, and sodium acetate and glycogen  were added to the aqueous phase at a final concentration of 496 mM and 0.17 mg/mL, respectively. The nucleic acid was then ethanol precipitated, resuspended in nuclease-free water, and quantified by NanoDrop 2000c (Thermo Scientific).w/v) agarose gels at 120 V for 45 min in 1× TAE  at room temperature. Gels were stained with ethidium bromide for 1 min and visualized using UV illumination. Two independent replicates were performed.To identify nucleic acid, 1× DNase buffer  was added to ~300–400 ng nucleic acid followed by the addition of either 2 units of RNase-free DNase I (Ambion) or 100 µg of DNase-free RNase A and incubation at 37 °C for 10 min. Treated samples and the untreated control were electrophoresed on 0.8% , 20 mM Tris-HCl (pH 8.0), 2 mM potassium phosphate (pH 7.5), 60 mM NaCl, 150 mM potassium glutamate, 4 mM magnesium acetate, 0.52 mM EDTA, 0.14 mM DTT, 0.1 mg/mL Bovine Serum Albumin (BSA), 0.004% (v/v) Triton X-100, and 30% (v/v) glycerol. In the case of the oligonucleotide ProU-40, the solution contained 40 mM Tris acetate (pH 7.9), 20 mM Tris-HCl (pH 8.0), 20 mM Tris-HCl (pH 7.6), 70 mM NaCl, 150 mM potassium glutamate, 4 mM magnesium acetate, 2 mM magnesium chloride, 0.5 mM EDTA, 0.14 mM DTT, 0.1 mg/mL BSA, 0.004% (v/v) Triton X-100, and 20% (v/v) glycerol. Solutions were incubated for 10 min at 37 °C, collected on ice, and electrophoresed on 12% (w/v) polyacrylamide  gels in 1× TBE at 150 V for 3 h at room temperature. Gels were visualized by autoradiography, and the films were scanned using a GS-800 calibrated densitometer (Bio-Rad). Quantification was performed using Quantity One software (Bio-Rad). Kd(app) was determine as the protein concentration needed to shift 50% of the 32P-labeled DNA.EMSAs with oligonucleotides (10 µL) contained the indicated amount of MotB, 0.01 pmol of v/v) Triton X-100, and 30% (v/v) glycerol at 37 °C for 10 min. Samples were electrophoresed on 0.8% (w/v) agarose gels at 70 V for 90 min in 1× TAE at 4 °C. Gels were stained with ethidium bromide for 1 min and visualized using UV illumination.For gel shift assays with T4 or λ DNA, 60 pmol MotB-His and/or H-NS were incubated with 500 ng of either T4 DNA treated with SspI or λ DNA treated with HindIII in a reaction mixture (5 µL) containing 20 mM Tris-HCl (pH 8), 2 mM potassium phosphate (pH 7.5), 60 mM NaCl, 0.42 mM EDTA, 0.04 mM DTT, 0.004% (w/v) polyacrylamide , 7 M urea denaturing gels for 2500 volt-h in 0.5× TBE. After electrophoresis, gels were visualized by autoradiography and analyzed as described above.Solutions were assembled in a total volume of 10 µL using 0.005 μM DNA (1 μL) and 0 to 3.2 μM of MotB (4 μL), as indicated, in the same buffer used for gel shift assays with oligonucleotides as described above. After 10 min of incubation at 37 °C, 0.8 units of DNase I (1.1 μL) was added to the reaction. Solutions were incubated for an additional 45 s at 37 °C and quenched with equal volumes of phenol. DNA was extracted, ethanol precipitated, and electrophoresed on 5% (BL21(DE3)/pLysE containing pTXB1-MotB or pTXB1 was grown in LB containing 25 μg/mL chloramphenicol and 100 μg/mL carbenicillin at 25 °C. MotB was isolated as described in Method I except that after binding to the chitin column, the resin was washed with 5 column volumes of CB buffer containing 500 mM NaCl followed by 5 column volumes of CB buffer containing 1 M NaCl. Proteins, isolated from the SDS-PAGE gel, were analyzed by mass spectrometry .600 ~0.1, grown to early log phase (OD600 ~0.3), and then transferred to ice until ready to titer. For each titer, ~50–100 phages in 100 μL LB were incubated with 200 μL of culture for 5 min at room temperature. Following addition of 2.5 mL 0.65% (w/v) Drake top agar at 45 °C [w/v) LB agar (Sigma) and incubated at 37 °C overnight. For infections using cells containing pNW129-MotB or pNW129, cultures were grown to an OD600 ~0.4 as described above and then protein synthesis was induced by the addition of 0.1% (w/v) arabinose  for 20 min before collecting on ice.Overnight cultures were diluted in fresh LB to an ODat 45 °C , cells w600 ~0.5). The NapIV S culture was transferred to ice for use as the plating strain. NapIV NS (900 μL) was transferred to a 1.5 microcentrifuge tube containing 100 μL of phage (MOI of ~0.004) pre-warmed at 37 °C. At 10 min post-infection, the phage-culture sample was diluted 10,000-fold in LB warmed to 37 °C with gentle vortexing to mix and returned to 37 °C. For pre-burst (≤30 min) and post-burst (35 to 70 min) time points, 100 μL or 10 μL of the diluted phage culture, respectively, were added to 100 μL of NapIV S followed by the addition of 3 mL of 0.65% Drake top agar (at 50 °C). The cells were immediately plated on 1.5% (w/v) LB agar and incubated overnight at 37 °C. Burst size was calculated by dividing the average number of plaques for the post-burst time points (50 to 70 min) by the average number of plaques for pre-burst time points (15 to 30 min).Burst size experiments were performed as described  for sing8 cells/mL (OD600 ~0.4) and then infected with either T4 wt or T4 ammotB at a MOI of 10. As indicated, RNA was isolated at 1, 5 or 10 min post-infection using method II as described [w/v) agarose gel stained with ethidium bromide.NapIV NS were grown to a cell density of ~4 × 10escribed . Brieflyescribed . RNA userRNA subtraction was performed with the bacterial RiboMinus Kit (Ambion) according to manufacturer instructions. cDNA was prepared using the NEBNext strand specific kit (New England BioLabs) according to manufacturer instructions for libraries with 300–450 bp insert size with the following modifications. Illumina adaptor sequences based on TruSeq HT Sample Prep Kits were purchased from Integrated DNA Technologies and used in the ligation step. TruSeq-1 and TruSeq-2 primer were used for PCR enrichment of adaptor ligated DNA. Library size was verified with a Bioanalyzer using an Agilent High Sensitivity DNA kit (Agilent Technologies). The concentration of each library was determined using the KAPA Library Quantification Kit  for Illumina platforms. Sequencing was performed by the NIDDK Genomics Core facility using a MiSeq system with the MiSeq 2 × 250 bp Sequencing Kit .Quality trimming (max ambiguity = 2) and trimming of 15 nucleotides from the 5’ terminal of all sequences were performed with CLC Genomics Workbench . The quality of raw sequencing data was accessed in CLC Biomedical Workbench software (Qiagen). A bimodal distribution of GC-content was observed in the data set with peak maximums at 36% GC and 55% GC . This diE. coli reference genome (NC_012971.2) and an unmapped reads file was generated. Approximately 44–54% of total reads mapped to the host genome. A significant number of host-encoded transcripts is consistent with other transcriptome analysis of bacteriophage infections during early and middle stages of temporal expression [ammotB. Statistical analysis was performed in CLC Genomics Workbench using the Baggerley’s Test with False Discovery Rate (FDR) corrected p values. RNA-seq data is available in the NCBI database (GEO number GSE113425) and in Trimmed data were aligned to the ome NC_0171.2 and pression . The unmCt method using CFX manager software (Bio-Rad), also known as 2Ct−ΔΔ [frd.2, nrdD, and motA, whose expression was unaffected in RNA-seq analysis, were used as endogenous controls. Primers were designed using Primer3web [RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (BioRad), and target genes were amplified with iTaq Universal SYBR green supermix (BioRad) in a BioRad CFX95. The relative gene expression was determined with the comparative s 2−ΔΔCt . Genes fimer3web ,33; sequ32P-labeled primer for each analysis. Primer extension products were separated on 5% (w/v) denaturing, polyacrylamide  gels. Gels were visualized by autoradiography and analyzed as described above.Primer extension was performed using Avian Myeloblastosis Virus (AMV) reverse transcriptase  as previously described ,35,36. OmotB, which had been cloned downstream of the arabinose-inducible promoter PBAD, in the E. coli B strain derivative BL21(DE3) were prevented by plasmid mutation(s) that eliminated the expression of motB. To mitigate this issue, all overnight cultures included low levels of glucose to reduce leaky expression from the PBAD promoter. To assess MotB toxicity, induction of MotB synthesis was done in both BL21(DE3) and in the E. coli K12 strain derivative TOP10F’ by the addition of arabinose. In BL21(DE3), a rapid reduction in cell density, as monitored by the OD600, was observed approximately 90 min after induction  was previously found as part of a prereplicative DNA complex after T4 infection , again sE. coli, whereas MotB-His retained comparable toxicity  for the T4 late gene 8, whose expression decreased 2.1-fold in the ammotB infection 5 min post-infection as detailed below K’s of 0.04 to 0.1 µM. It should be noted a comparison of MotB vs. MotB-His binding  to bind different dsDNA oligonucleotides: (1) a 37 bp fragment containing positions −29 to +8 of the promoter  a ProU-40, c; and K’s , MotB affinity for the 20 bp fragment decreased ~10-fold, suggesting that a fragment size greater than ~20 bp is needed for optimal binding. Finally, we also tested the ability of MotB-His to bind ssDNA, using the 37 nucleotides top strand of the ds P8 fragment  arabinose for only 20 min before plating on LB agar containing no arabinose, thereby producing a low level of MotB prior to infection. In fact, the level of produced MotB was too low to be detected by SDS-PAGE. Nevertheless, prior production of MotB resulted in larger plaques when infected with T4 wt, T4 ammotB, or T4 ammotA  by the motB knock-down  were unchanged. We conclude that transcription of late genes 7, 8, 15, 20 and 27, which is lower at 5 min in the T4 ammotB infection, is fully restored by 10 min.Using the 5 min RNA, we were unable to quantify the levels of all the differentially expressed late RNAs because the level of late transcription at this time is so low. However, with Pls tested . In partmotB altered the overall transcription pattern. As seen in Finally, we used primer extensions within a specific early, middle, or late region at 1, 5 or 10 min post-infection, respectively, to investigate whether the absence of motB, located in a large dispensable region of the T4 genome, called Δ12  [E. coli [motB when it is heterologously expressed in E. coli and the phage burst increase in its presence lead to the conclusion that MotB is needed for optimal phage growth, perhaps through a host takeover function.Although many aspects of bacteriophage T4 biology have been extensively characterized, dozens of T4’s ~300 genes encode proteins whose specific functions remain unknown. Most of these are nonessential genes that are expressed early, leading to the presumption that they are helpful for host takeover, particularly under certain conditions. In this study, we have characterized one such early gene, to 3778) . Other g[E. coli ,43. The motB in a motA knockdown infection [ammotB infection. Rather only the expression of a few late genes is lowered at 5 min post-infection. By 10 min when late transcripts are highly expressed, transcription of these genes has recovered. Therefore, it seems unlikely then that MotB directly regulates T4 gene expression unless this function is redundant within the genome.Previous work indicated that MotB can be isolated in a pre-replicative complex containing DNA, RNAP, and other polymerase-associated and DNA-binding proteins . Other wnfection . Taken tammotB infection. Instead, we observe MotB protection of the entire region of the DNA once a sufficient protein concentration is reached. This type of binding profile is indicative of a protein that can “spread” along the DNA with little, if any sequence specificity, perhaps through protein–protein cooperativity. However, we cannot eliminate the possibility that MotB has a high affinity site that was not present in the tested DNAs.Our biochemical analyses indicate that MotB is a protein that binds to both unmodified and T4 wt GHme-modified DNA. It binds very tightly to host DNA when produced heterologously in vivo, it binds with ~100 nanomolar affinity to various ds oligonucleotides, and in EMSAs it retards each of the λ/HindIII fragments. The DNase I footprint of MotB on an unmodified 218 bp fragment, which includes the T4 late promoter P8, does not reveal a high affinity binding site despite the down-regulation of late gene 8 in the T4 E. coli histone-like proteins that also can spread along the DNA with limited DNA specificity. H-NS, and its less abundant homolog StpA, target AT-rich DNA sequences, condensing genomic DNA through the formation of ordered and looped structures, and typically repress transcription at the affected region . Our gemotB is highly toxic, resulting in rapid cell lysis of BL21(DE3) cells and growth arrest of TOP10F’ cells. The bactericidal properties of phage proteins when expressed in their host is one of the driving forces of phage research. The mechanisms by which proteins such as MotB inhibit bacterial growth could provide insight for the development of future antibiotics.Given the high predicted pKa of MotB (9.23) and its presence in the T4 dispensable region Δ12, we can determine its relative abundance during infection from previous 2-D gels of radiolabeled T4 proteins at various times after infection . We iden


Escherichia coli. However, the recombinant PBPs are incompletely chromophorylated, and the underlying mechanisms are not clear.Phycobiliproteins (PBPs) are light-harvesting protein found in cyanobacteria, red algae and the cryptomonads. They have been widely used as fluorescent labels in cytometry and immunofluorescence analysis. A number of PBPs has been produced in metabolically engineered E. coli was constructed using a single-expression plasmid strategy. Compared with a previous E. coli strain transformed with dual plasmids, the E. coli strain transformed with a single plasmid showed increased plasmid stability and produced SLA-PEB with a higher chromophorylation ratio. To achieve full chromophorylation of SLA-PEB, directed evolution was employed to improve the catalytic performance of lyase CpcS. In addition, the catalytic abilities of heme oxygenases from different cyanobacteria were investigated based on biliverdin IXα and PEB accumulation. Upregulation of the heme biosynthetic pathway genes was also carried out to increase heme availability and PEB biosynthesis in E. coli. Fed-batch fermentation was conducted for the strain V5ALD, which produced recombinant SLA-PEB with a chromophorylation ratio of 96.7%.In this work, a pathway for SLA-PEB [a fusion protein of streptavidin and allophycocyanin that covalently binds phycoerythrobilin (PEB)] biosynthesis in In addition to reporting the highest chromophorylation ratio of recombinant PBPs to date, this work demonstrated strategies for improving the chromophorylation of recombinant protein, especially biliprotein with heme, or its derivatives as a prosthetic group.The online version of this article (10.1186/s12934-019-1100-6) contains supplementary material, which is available to authorized users. The phycobiliproteins (PBPs) are a family of light-harvesting proteins found in cyanobacteria, red algae and the cryptomonads . Based o−13 [PBPs are generally extracted and purified from cyanobacteria and red algae . When us−13 . The strSynechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, CpcS/U is composed of a heterodimer of two similar proteins, designated CpcS-I and CpcU [Nostoc sp. PCC 7120, the CpcS lyase exists in a monomeric, single subunit form [Escherichia coli [E. coli. The fusion protein with PEB as chromophore was successfully produced in E. coli. While retaining the fluorescent properties, the fusion protein could bind biotinynated antibody and could be used as fluorescent label in immunofluorescence assay [In recent decades, pathways for the most abundant PBPs in cyanobacteria, APC (ApcA/ApcB) and PC (CpcA/CpcB), have been elucidated. Phycocyanobilin (PCB) and phycoerythrobilin (PEB) are the major phycobilins attached to APC and PC. These phycobilins originate from cyclic tetrapyrrole (heme). The oxidative cleavage of heme by heme oxygenase (Ho1) produces biliverdin IXα (BV), the first intermediate common to all phycobilins. BV is subsequently reduced by ferredoxin-dependent bilin reductases to yield PCB, PEB or other types of phycobilins . The attand CpcU , 10, whinit form . The thihia coli –16. Thesce assay .E. coli, however, were partially chromophorylated. The chromophorylation ratio of PBPs  was reported to range from 17.4 to 71.9% [E. coli. The apo-PBPs are difficult to remove from the PBP mixture during protein purification and lead to low detection sensitivity in immunofluorescence assays due to the lack of fluorescence emission. It was speculated that the incomplete chromophorylation is due to unfavorable codon usage that limits the expression of large amounts of lyases and phycobilins by E. coli, or it is due to aggregation of the recombinant proteins into insoluble inclusion bodies [The recombinant PBPs produced in to 71.9% , 17, 18,n bodies . In addin bodies . SpectraE. coli. It was found that the E. coli strain transformed with dual expression plasmids lost one or both plasmids during the course of fermentation. To improve plasmid stability, we reconstructed the biosynthetic pathway in E. coli by using one expression vector. This strain, which has increased plasmid stability, retained the entire SLA-PEB biosynthetic pathway and produced recombinant SLA-PEB with a higher chromophorylation ratio. To achieve full chromophorylation, the catalytic abilities of Ho1 from different cyanobacteria were investigated based on BV and PEB accumulation. Upregulation of the heme biosynthetic pathway genes was also carried out to increase heme availability and PEB biosynthesis in E. coli. These strategies enable the efficient production of SLA-PEB in E. coli. In a 5 L fermentor, the engineered strain V5ALD produced 56.4 mg/L SLA-PEB with a chromophorylation ratio of 96.7%.In this study, we tried to elucidate the mechanisms underlying the incomplete chromophorylation of recombinant SLA-PEB and construct an efficient pathway for SLA-PEB production in E. coli strain for SLA-PEB biosynthesis was constructed in our previous work [SLA-cpcS and pRSF-PHo1-pebS. The fusion protein SLA-PEB with 6× His tag at its N-terminal can be easily purified by Ni2+ chelating affinity chromatography. To express the SLA-PEB pathway genes from one expression vector, PHo1 and pebS (coding phycoerythrobilin synthase) were designed as a polycistron. This polycistron was chemically synthesized and ligated into the second expression cassette of the vector pRSFDuet-1, generating the plasmid pRSF-PHo1-pebS. The BamHI/SacI fragment of SLA from pCDF-SLA-cpcS was ligated into similarly digested pRSF-PHo1-pebS, generating the plasmid pRSF-SLA-PHo1-pebS. cpcS was amplified by PCR using primers cpcSF and cpcSR with a ribosomal binding site engineered on the primer cpcSF. The PCR product was digested with SacI/SalI, purified and ligated into similarly digested pRSF-SLA-PHo1-pebS, generating the expression plasmid pRSF-SLA-cpcS-PHo1-pebS , generating the strain SLA-V2 , Synechococcus elongatus BP-1(BHo1), Synechococcus sp. PCC 9311(9Ho1), Synechocystis sp. PCC 6803(6Ho1) and cyanophage PSSM-2(PHo1) were optimized according to E. coli codon usage and artificially synthesized. These Ho1 genes were digested with NdeI/BglII and cloned into similarly digested pRSFDuet-1, generating the plasmids pRSF-7Ho1, pRSF-BHo1, pRSF-9Ho1, pRSF-6Ho1 and pRSF-PHo1. These plasmids were individually transformed into E. coli BL21(DE3), generating the BV-producing strains EBV7, EBVB, EBV9, EBV6 and EBVP. The pebS gene was amplified using the primers pebSF and pebSR, digested with BglII/KpnI and ligated into similarly digested pRSF-7Ho1, pRSF-BHo1, pRSF-9Ho1, pRSF-6Ho1 and pRSF-PHo1, generating the plasmids pRSF-7Ho1-pebS, pRSF-BHo1-pebS, pRSF-9Ho1-pebS, pRSF-6Ho1-pebS and pRSF-PHo1-pebS, respectively. These plasmids were individually transformed into competent E. coli BL21(DE3), generating the PEB-producing strains PEB7, PEBB, PEB9, PEB6 and PEBP.NdeI/BglII fragments of Ho1 were ligated into NdeI/BglII digested pRSF-SLA-cpcSM7-PHo1-pebS, generating the plasmid pRSF-SLA-cpcSM7-BHo1-pebS, pRSF-SLA-cpcSM7-6Ho1-pebS, pRSF-SLA-cpcSM7-7Ho1-pebS, pRSF-SLA-cpcSM7-9Ho1-pebS, respectively. These plasmids were individually transformed into E. coli BL21(DE3), generating the strains SLA-V4B, SLA-V46, SLA-V47, SLA-V49, respectively.The hemL (from E. coli) and hemAS  transformed with plasmid pKD46 [E. coli strains EAL, EALB, EALD, EALF, ELAG and EALH. Integration of the expression cassettes was confirmed by PCR using gene-specific primers and chromosome-specific primers  were deKOarsBCm , generatid pKD46 , generatcpcS mutant library. The plasmid pCDF-SLA-cpcS at a concentration of 5 ng/μL was used as the DNA template, and cpcSMF and cpcSMR were used as the primers  was used to generate a Escherichia coli BL21(DE3) was transformed with the plasmid library. The transformants were spread on LB agar plates . The LB agar plates contained 1.0 mM ALA and 1.0 g/L lactose, and were cultivated at 18 °C for 96–120 h in darkness. Positive colonies were selected according to color change. The detailed procedure is described in Additional file E. coli cells was extracted with acetone overnight in darkness. The extractions were filtered through 0.45 μm polytetrafluoroethylene syringe filter and then were resolved by reversed-phase chromatography using an Agilent Technologies 1260 system. The HPLC column was a 4.6 × 250-mm Phenomenex Ultracarb analytical column. The mobile phase consisted of acetone and 20 mM formic acid (50:50 by volume), and the flow rate was 0.6 mL/min. The eluates were monitored at 650, 560, and 380 nm using an Agilent Technologies 1260 series diode array detector. As needed, complete spectra were obtained for the peaks desired. Peak areas were quantified using Agilent Technologies Chemstation software.For the analysis of BV and PEB, the method described by Frankenberg was basically followed . The BV E. coli cultivation for the DNA manipulations and seed cultures. For expression of foreign proteins, TB medium was used, which contains 12 g/L tryptone, 24 g/L yeast extract, 4 g/L glycerol, 2.31 g/L KH2PO4, 12.54 g/L K2HPO4. Kanamycin  and/or spectinomycin  were added to culture medium to provide selective pressure. Single colonies of recombinant E. coli were grown in 3 mL LB medium. 15 mL of overnight seed cultures were then inoculated into 300 mL of TB medium containing the appropriate antibiotics and cultured at 37 °C until the optical density at 600 nm reached 0.8. The cultures were then cooled to 18 °C, and the expression of the foreign genes was induced by the addition of isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.1 mM. After a further incubation for 24 h at 18 °C in the dark, the E. coli cells were harvested by centrifugation at 6000×g for 10 min.LB medium was used for 4·H2O, 7 g/L KH2PO4, 8 g/L K2HPO4. The culture was cooled to 18 °C when the cells were in the mid-exponential stage. The cells were then induced with 0.5 mM IPTG and cultivated for 18 h.The fed-batch fermentation was performed in a 5 L fermentor . 2.0% inoculation of overnight seed culture was transferred into the fermentor with 2.5 L TB medium. The constant feeding mode (0.01 g/L/min) was employed to achieve high cell density. The feeding medium was composed of 200 g/L gluocose, 5 g/L NaCl, 5 g/L MgSOg for 30 min at 4 °C, the supernatants were loaded onto a pre-equilibrated column of Chelating Sepharose  charged with Ni2+. The column was then washed with washing buffer  to remove weakly bound proteins. The fusion proteins were eluted with elution buffer . The high-concentration imidazole was removed using a Sephadex G25 column. The peak fraction was collected and concentrated by ultrafiltration. Protein concentrations were measured by the dye-binding method of Bradford using a commercial kit .The cells were suspended in 30 mL of binding buffer  and disrupted by sonication for 30 min on ice. After centrifugation at 6000×E. coli was analyzed by SDS-PAGE. E. coli cells expressing Ho1 were disrupted by sonication and then centrifugated at 6000×g for 30 min at 4 °C. The supernatant was mixed with loading buffer and boiled for 10 min. 10 μL protein samples were loaded on 12% acrylamide gel. Resolved proteins were visualized by staining with Coomassie Blue.Expression of Ho1 in Absorption spectra were obtained using a UV-1801 spectrophotometer  with a 0.5 cm path length cuvette. The wavelength range was from 250 to 700 nm, and the sample interval was set to 1 nm with a scan speed of 240 nm/min.The fluorescence emission spectra were analyzed using an F-4500 fluorescence spectrophotometer . The excitation wavelength was 520 nm, and the emission spectra were recorded from 540 to 610 nm. The emission and excitation slit widths were set to 5 nm with a scan speed of 240 nm/min.−1 cm−1 at 560 nm. To calculate the chromophorylation ratio, the concentration of PEB was divided by the concentration of purified recombinant proteins.The recombinant proteins were denatured in 8.0 M urea (pH 1.5). The PEB concentrations were calculated using an excitation coefficient of 42.8 mMThe intracellular heme concentration was determined according to the procedure described by Sassa with slight modification , 25. TheThe plasmid stability for the strains SLA-V1 and SLA-V2 were compared at the end of expression period. SLA-V1 was grown in TB medium containing 100 μg/mL Sm and 100 μg/mL Km, while SLA-V2 was grown in TB medium containing 100 μg/mL Km. To evaluate the effect of IPTG induction on plasmid stability in these strains, these cultures were induced with or without IPTG at a final concentration of 0.1 mM. One hundred microliter samples were taken from flasks at the end of the 24 h expression period. Each sample was diluted in LB medium and spread on nonselective LB agar plates. These plates were incubated at 37 °C for 16 h. Individual colonies were randomly chosen and replica-plated on various selective LB plates containing 100 μg/mL Sm, 100 μg/mL Km, or 100 μg/mL Sm and 100 μg/mL Km. The plasmid stability was defined as the fraction of plasmid(s)-bearing cells and was determined by taking the ratio of the number of colonies on the selective plates to the total number of transferred colonies.To determine plasmid structural stability, colony PCR amplification using primers duetup1/duetup2 and T7 terminator was carried out to analyze the stability of expression cassettes.E. coli strain SLA-V1 was constructed in our previous study to produce SLA-PEB. In this strain, the fusion protein (SLA) of core streptavidin from Streptomyces avidinii and allophycocyanin alpha subunit from thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, together with lyase CpcS from Thermosynechococcus elongatus BP-1 and PEB producing enzymes (PHo1 and PebS) from cyanophage PSSM-2, were co-expressed in E. coli. Two plasmids for were used for the expression of the four genes required for SLA-PEB biosynthesis [E. coli [E. coli using dual or multiple plasmid expression systems [E. coli is an important factor affecting the chromophorylation of recombinant SLA-PEB.The engineered ynthesis . The rec[E. coli , 14, 18. systems , 14, 19. systems –28. We sE. coli cells could not grow on selective plates, indicating plasmid instability in the E. coli cells. Under uninduced conditions, the E. coli cells had high plasmid stability. Only 1.9% of the E. coli cells lost the plasmid pCDF-SLA-cpcS. Under induced conditions, 16.2% of the E. coli cells lost the plasmid pCDF-SLA-cpcS, 10.6% of the induced E. coli cells lost the plasmid pRSF-PHo1-pebS, and 6.9% of the induced E. coli cells lost both plasmids. Only 66.3% of the induced cells retained both expression plasmids , generating the strain SLA-V2. Under uninduced conditions, no E. coli cells lost the expression plasmid. Under induced conditions, 5.2% of the E. coli cells lost the expression plasmid  limiting the chromophorylation of recombinant SLA-PEB. Both PEB depletion and low activity and/or expression level of CpcS would cause incomplete chromophorylation. It was shown by HPLC analysis that PEB accumulated in SLA-V2 cells . HPLC analysis showed that in SLA-V3 cells neither PEB nor BV could be detected , the chromophorylation ratio of recombinant PBPs was remarkably decreased, though its production level was elevated (data not shown). To examine whether the strain V5ALD had superior performance in PBP production, both E. coli strains were cultivated in a 5 L fermentor. IPTG was added to the culture medium at a final concentration of 1.0 mM when the cell density reached an OD600 of 8.0. Constant feeding mode was employed to achieved high cell density. While both strains had similar cell densities at the end of the fermentation, strain V5ALD produced a higher level of SLA-PEB (56.4 mg/L) than the strain SLA-V1 (32.5 mg/L). Most importantly, the chromophorylation ratio of the SLA-PEB purified from V5ALD was 96.7%, which was much higher than that from strain SLA-V1 (Fig. We noticed that when E. coli leads to the production of a mixture of holo-PBP and apo-PBP. In this study, the biosynthesis pathway for phycobiliprotein was constructed using one expression plasmid, which had significantly increased plasmid stability. Moreover, strategies for the directed evolution of lyase, the upregulation of heme biosynthetic pathway genes and for overcoming the limiting factors in PEB biosynthesis were employed to enhance the production level and chromophorylation of recombinant phycobiliprotein in E. coli. A chromophorylation ratio of 96.7% for SLA-PEB was achieved in fed-batch fermentation in this study, and this value was the highest ever reported from an engineered E. coli strain. This study demonstrates the validity of a single-expression vector strategy and metabolic engineering for establishing an efficient biosynthetic pathway for PBP production in E. coli.An ideal cell factory should demonstrate high efficiency of target product biosynthesis. Inefficient biosynthesis of recombinant PBPs in recombinant Additional file 1: Table S1. Primers used in this study. Table S2. Recombinant E. coli strains used in this study. Figure S1. Flowchart of directed evolution of CpcS for improving chromophorylation efficiency of SLA-PEB. Figure S2. Colony PCR for detection of the presence of pCDF-SLA-cpcS and pRSF-PHo1-pebS in E. coli strain SLA-V1. The primers are duetup-2 and T7 terminator. The expression cassette of cpcS in pCDF-SLA-cpcS is 813 bp, and the expression cassette of Ho1 and pebS is 1709 bp. DNA marker: 8000 bp, 5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp and 100 bp. Figure S3. Colony PCR for detection of the stability of expression cassette. The primers are duetup-1 and T7 terminator. The size of expression cassette of SLA, CpcS, PHo1 and pebS is 3433 bp. DNA marker: 8000 bp, 5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp and 100 bp. Figure S4. HPLC analysis of PEB extracted from E. coli strains. A: SLA-V2; B: SLA-V3; C: SLA-V4B. Figure S5. SDS-PAGE analysis of Ho1s expression in E. coli strains. 1: EBV7, 2: EBVP, 3: EBV6, 4: EBV9, 5: EBVB. The calculated molecular weight is 27.1 kDa for 7Ho1, 26.9 kDa for PHo1, 27.0 kDa for 6Ho1, 26.7 kDa for 9Ho1 and 27.1 kDa for BHo1.


We confirmed that our protocol can also be applied to purify other varied AAV genome constructs. Our protocol can scale up production of pure rAAV9, in compliance with current good manufacturing practice, for clinical applications in human gene therapy.Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically transduce muscle and neuronal tissues; thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable. We recently described a chromatographic purification protocol for rAAV1; this protocol can achieve scalable purifications. In this study, we attempted to optimize this protocol for purifying rAAV9 preparations, and we developed a novel, effective method for high-yield purification of rAAV9 using quaternary ammonium anion exchangers and size-exclusion chromatography. The final purified rAAV9 contained mainly three capsid proteins, as observed by SDS-PAGE. Furthermore, negative-stain electron microscopy demonstrated that 96.1% ± 1.1% of rAAV9 particles carried the viral genome containing the EGFP transgene, indicating that impurities and empty capsids can be eliminated with our purification protocol. The final rAAV9 titer obtained by our protocol totaled 2.5 ± 0.4 × 10 Thus, collecting rAAVs secreted in the culture media, which exclude serum, and their chromatographic purification are applicable to optimal purification of most AAV serotypes, resulting in high purity and high yields.Chromatographic purification is more suitable for large-scale vector purification than commonly used ultracentrifugation methods. Thus, several chromatographic protocols have been proposed for purifying rAAVs.E1 gene region  and the BCL-XL gene)cis, trans, and helper plasmids to produce high yields of rAAV9. Most of the contaminants and empty capsids in the culture supernatant can be separated from purified rAAV9 by this easy purification method. Thus, our simple protocol is capable of producing high-quality rAAV9 with a high manufacturing yield.Among the AAV serotypes, rAAV9 is expected to be used widely in human gene therapy because of its broad tissue tropism. For instance, rAAV9 can transduce the cardiac muscles more efficiently than serotype 8.Previously, we successfully used ammonium sulfate (AS) to precipitate and concentrate rAAV1.E1 gene region  and the BCL-XL gene; these cells yield 2-fold more rAAV than HEK293 cells.9 HEK293EB cells . After 1/3→1/2 AS treatment, the AAV9-dsEGFP sample was dissolved in 20 mL of 3.3 mM morpholinoethanesulfonic acid, 3.3 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 3.3 mM sodium acetate buffer  containing 50 mM NaCl and 0.01% (w/v) Pluronic F-68. This purification method was based on the results of a preliminary small-scale experiment (15 v.g. or 3.7 × 1014 vector genomes (v.g.), measured by qPCR using primers targeting the inverted terminal repeats (ITR) or EGFP, and the final product contained 3.8%  of empty capsids, as determined by negative-stain electron microscopy (EM)   primers (GAPDH (detection limit of one copy of human GAPDH) was detected in the final product (containing 1.0 × 109 v.g.) and in the solvent of the final product; however, 2.6 copies of human GAPDH were detected in the post-tangential flow-filtration (post-TFF) solvent (containing 1.0 × 109 v.g.). Therefore, human genomic DNA contamination can be removed with the chromatographic purification process described here. Furthermore, we evaluated the efficiency of the purified AAV9-dsEGFP to transduce HEK293EB cells  , or 3.7  primers . No copyEB cells . The thr15 v.g., as measured using ITR-targeted primers , and that of pure AAV9-ssLuc was 1.8 × 1014 v.g., as measured using ITR-targeted primers   and single-stranded AAV-CMV-RFLuc (AAV9-ssLuc) transgenes. The same purification procedure as used to purify AAV9-dsEGFP was performed. AAV9-dsLuc and AAV9-ssLuc preparations are shown in primers) . Thus, wWe recently developed an efficient protocol for rAAV1 preparation using chromatographic purification and excluding ultracentrifugation.in vivo transduction experiments. Potter et al.15 v.g., as measured using ITR-targeted primers , which is comparable with or higher than that obtained using other protocols. In our final preparations, 96.1% ± 1.1% of the particles contained the viral genome, according to our negative-stain EM analysis  were cultured at 37°C in a 5% CO2 atmosphere in DMEM  containing 8% (v/v) fetal bovine serum (FBS)  and penicillin and streptomycin . HEK293 cells were used for optimization of AS treatment conditions and in small-scale experiments. In laboratory-scale experiments, HEK293EB cells  were plated in 16 square culture dishes  with a surface area totaling 8,000 cm2. The initial volume of medium was 1,120 mL.HEK293 or HEK293EB cells . In pdsAAV-CBA-RFLuc, the red firefly luciferase (RFLuc) fragment was amplified by PCR using the pCMV-RedFluc plasmid  as a template and using the primers 5′-AACGAATTCGGATCCGCCACCATGGAAACAGAAAGAGAAGAAAACG-3′ and 5′-CTGGAATTCAAGCTTCTACCCACCTGCTTGAGGTTTCTTG-3′; the resultant PCR product was digested using NcoI and HindIII, and it was used to replace the EGFP in pdsAAV-CBA-EGFP. In pssAAV-CMV-RFLuc, the RFLuc fragment was amplified by PCR as described above and cloned into the EcoRI-HindIII site of pAAV-MCS (Agilent Technologies). The trans plasmid, pAAV2/9 (with the AAV type 2 rep gene and type 9 cap gene), was donated by James M. Wilson (University of Pennsylvania). The adenovirus helper plasmid, pHelper , was purchased from Stratagene .The three cis, trans, and adenovirus helper plasmids were transfected at a ratio of 1:1:2cis plasmid, 44.8 μg of trans plasmid, and 90.1 μg of pHelper per square dish) to cells maintained in DMEM without serum supplemented with GlutaMAX-І  and using polyethyleneimine  at a DNA:PEI ratio of 1:2.g for 10 min each. Next, 1/3 AS precipitation was performed by adding half of the sample volume of saturated AS to produce a final solution of 33% (w/v) AS, followed by centrifugation at 18,800 × g for 30 min at 4°C. rAAV9 was finally precipitated in 1/2 AS solution  (1/3→1/2 AS), followed by centrifugation at 18,800 × g for 30 min 4°C. The precipitated rAAV9 was dissolved in 20 mL of a buffer containing 3.3 mM morpholinoethanesulfonic acid , 3.3 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Dojindo), and 3.3 mM sodium acetate  (MHN buffer), pH 8.0, for laboratory-scale purification using a HiPrep Q XL 16/10 column . The MHN buffer contained 50 mM NaCl and 0.01% (w/v) non-ionic surfactant Pluronic F-68 (Sigma-Aldrich).HEK293 or HEK293EB cells were grown for 2 days to reach 90% confluence. The For laboratory-scale purification, 20 mL of 1/3→1/2 AS-treated sample was dissolved and diluted in MHN buffer until the conductivity of the solution decreased to 7.3 millisiemens/cm. The conductivity was measured using an electroconductivity sensor . The diluted sample was loaded onto a quaternary ammonium anion exchanger with a bed volume of 20 mL (HiPrep Q XL 16/10) equilibrated with each buffered solution at a rate of 3 mL/min and controlled by a P1 peristaltic pump . The pass-through fraction was collected and ultrafiltrated using Ultracel 30 K centrifugal filters . We also collected the sample washed out through the anion-exchange column and the sample eluted from the anion-exchange column using MHN buffer (pH 8.0) containing 1 M NaCl and 0.01% (w/v) Pluronic F-68. The pass-through fraction was purified by size-exclusion (gel filtration) chromatography using a HiLoad 16/60 Superdex 200 preparation-grade column  and an ÄKTA Explorer 100 HPLC system  equipped with a 10-mL sample loop running the MHN buffer (pH 6.5) containing 300 mM NaCl and 0.01% (w/v) Pluronic F-68. Fractions were collected as follows: first segment, 0.2 column volume (CV), 5-mL fractions; second segment, 0.6 CV, 1-mL fractions; third segment, 0.3 CV, 5-mL fractions (1 CV = 120 mL). Peak fractions (fractions 15–27) were collected to obtain the final product. The purity of the final rAAV9 product was assessed by 5%–20% (v/v) gradient gel SDS-PAGE  and protein staining with either Quick Coomassie brilliant blue  or Oriole fluorescent gel stain  and by western blotting using an anti-AAV-capsid monoclonal antibody .2 for 30 min at 37°C. The viral genome DNA was extracted using the DNeasy Blood & Tissue Kit . The benzonase-resistant genome titer was measured by real-time qPCR  using ITR-targeted primers ,For purification of rAAV viral DNA, purified rAAV9 was treated with 125 U/mL benzonase and 2 mM MgClThe purified rAAV9 preparation was separated by 5%–20% (v/v) gradient polyacrylamide gel (Wako) in a running buffer containing SDS. To visualize and analyze the SDS-PAGE bands during AS treatment condition optimization and small-scale experiments, we used Q-CBB (Wako), and for the rest of the experiments we used Oriole fluorescent gel stain (Bio-Rad). The sensitivity of the Oriole gel stain is almost equal to that of silver staining.2, and then the viral DNA was purified with the DNeasy Blood & Tissue Kit. For detection of contaminating human genomic DNA outside of rAAV9 particles, the viral DNA was analyzed without benzonase treatment. The qPCR conditions were 10 s at 95°C, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s.Contaminating human genomic DNA in final preparations  was quantified by qPCR using human GAPDH-targeting primers 5 HEK293EB cells were infected with purified AAV9-dsEGFP at 5.0 × 106 v.g./cell. Three days after transduction, the proportion of EGFP-positive cells was measured by flow cytometry  with propidium iodide staining  to identify and exclude nonviable cells. Significant differences were determined using ANOVA by Microsoft Excel.Triplicate wells containing 4.7 × 10A carbon-stabilized copper grid  was placed onto a test sample and incubated for 1 min. The sample on the grid was then negatively stained for 1 min with 1% (w/v) uranyl acetate. Finally, the grid was examined using a H-7650 transmission electron microscope  at an accelerating voltage of 80 kV. The ratio of empty particles to encapsulated particles was determined by counting the number of packaged and unpackaged particles in the electron micrographs.Sedimentation velocity AUC analysis was conducted with a Proteome Lab XL-1 ultracentrifuge . Four hundred microliters of AAV9-dsEGFP was loaded into the sample compartment of the centerpiece, and 400 μl of solvent was loaded into the reference compartment of the centerpiece. The four-hole rotor loaded the sample in the instrument, and it was equilibrated to a temperature of 20°C. Sedimentation velocity centrifugation was performed at 12,000 rpm and 20°C, and absorbance (260 nm) was used for analysis.F statistic, 0.68; buffer density, 1.0148; buffer viscosity, 0.0106. This model fits the data to the Lamm equation. The reliability of the analysis was confirmed by the goodness of fit (root-mean-square deviation [RMSD]).The percentages of fully packaged virions, intermediate particles, and empty particles were determined by analyzing 92 scans and the Sedfit continuous-size C(S) distribution model. The following C(S) parameters were used for analysis: resolution, 100 S; S min, 20; S max, 120; frictional ration, 1.32859; T.T. designed the experiments, performed most of the experiments, analyzed data, and wrote the manuscript. H.O. and Y.M. conducted the experiments, analyzed data, and wrote the manuscript. K.A. performed some experiments. S.S., Y.K., H.C., and J.M. made experimental suggestions. A.I., M.O., and A.T. supervised the project. Y.H., T.S., and T.O. designed the experiments, conducted the experiments, analyzed data, and wrote the manuscript. All authors read and approved the manuscript.The authors have no conflicts of interest.