June 2015 Wednesday, June 24 ● Outlined overall project goals ● Used NCBI’s nucleotide best BLAST to find gene orthologs from the Garcia pathway in H. volcanii. ○ Found all genes in H. volcanii except for butyraldehyde dehydrogenase and butanol dehydrogenase. ● Used BLAST to find other butyraldehyde and butanol dehydrogenases that could be used in H. volcanii. ○ Considered using an aldehyde/alcohol dehydrogenase fusion gene. ● Found a promising fusion gene in Halomonas sp. S2151 called adhE2. Also had a pI near 5, which is ideal for H. volcanii. ○ Little was known about Halomonas and the gene was only recently discovered. ● Considered using gas chromatography to confirm the presence of butyryl-CoA. Thursday, June 25 ● Continued to pursue the idea of using gas chromatography to test for butyryl-CoA. ○ Rob Franks agreed to allow us to use the GC. ● Searched for places to purchase butyryl-CoA to create a standard for GC, but it’s very expensive. ● Started searching for protocols for gas chromatography of butyryl-CoA and more generally, fatty acids. Friday, June 26 ● Created 10 L of 30% salt water stock to be used in producing media for H. volcanii. ○ Used protocol in pg 20 of Halohandbook. ● Prepared Tris buffer (500 mL of pH 8.83 and 40 mL of pH 7.54). Monday, June 29 ● Continued searching for gas chromatography protocols specific to butyryl-CoA. ● Considered using qPCR, since finding a protocol is a challenge. ● Sources suggest using HPLC to detect butyryl-CoA. ○ Rob Franks believes HPLC will be better suited for butyryl-CoA. ● Found a site with potentially helpful primer design rules (http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html) Tuesday, June 30 ● Decided to stop pursuing gas chromatography of butyryl-CoA. We are assuming that H. volcanii produces it at this point. ● MGM plates have been prepared according to pg 14 of Halohandbook. July 2015 Wednesday, July 1 ● Proposed the idea of using beads that bind to CoA groups to detect the presence of butyryl-CoA. ● Trying to find a good promoter to increase the gene expression of the genes we decide to use. ○ Several searches yielded the promsing ptb promoter. ● Considered using rational design to lower the pI of the fusion protein to near 4.5. Requires generating a model structure using SWISS-MODELLER. ○ Unable to find a 3D model of adhE2. ● Jairo used his program to codon optimize the adhE2 gene for H. volcanii. Thursday, July 2 ● Decided on the pTA963 plasmid for eventual expression of our fusion gene. ● Professor Bernick urged us to focus on pI rather than percent identity of potential fusion genes. ● Found yet another alternative fusion gene in Shewanella waksmanii (http://www.ncbi.nlm.nih.gov/nuccore/655366186). ○ Found the nucleotide sequence for this fusion gene. ○ Discontinuing work on the Halomonas fusion gene adhE2. Monday, July 6 ● Attempting to find a protocol for inserting the S. waksmanii fusion gene into the expression plasmid. ○ Dominic suggested using Gibson assembly. ● Tried to codon optimize the S. waksmanii gene using Jairo’s program, but it ran into a syntax error which couldn’t be fixed. ● Asked Professor Bernick to contact an expert in S. waksmanii (Chad Saltikov). ● Reconsidered the ptb promoter as a backup. ● Found the bgaH gene, which is used to assay the promoters of haloarchaeal genes. ● Found a similar fusion gene in Shewanella sediminis. ○ Has a lower pI. ○ Unable to find the entire genome of S. waksmanii for codon optimization. ○ The full genome for S. sediminis is available, allowing codon optimization. Tuesday, July 7 ● Professor Bernick approved the idea of using the S. sediminis fusion gene. ● The fusion gene was codon optimized for H. volcanii. ● Checked nucleotide sequence for the formation of structures that would affect Gibson assembly. ● Split the S. sediminis fusion gene into smaller 500 - 700 bp fragments for ordering. ○ Fragments >1000 bp take longer and are more expensive. Wednesday, July 8 ● Finished splitting the S. sediminis fusion gene into 3 gene fragments. ● Began designing primers for the pTA963 plasmid and the His-tags. Thursday, July 9 ● Plasmid primers were ordered. ● Added overlaps on the S. sediminis fusion gene for both N- and C-terminal His tags. Friday, July 10 ● Continuing to pursue the bgaH gene as a backup. ● Started working on primers for fusion gene. Saturday, July 11 ● Finished fusion gene primers. Monday, July 13 ● Waited for approval of S. sediminis primers for ordering. ● Ran inverse PCR on the pTA963 plasmid. Tuesday, July 14 ● Checked optimized gene sequence to ensure that resulting amino acid sequence matches. ○ Did not match, so the program needs refinement. Wednesday, July 15 ● Jairo attempted to fix codon optimization program. Thursday, July 16 ● Jairo fixed his program, however Professor Bernick urged us to use DNAWorks for codon optimization. ● Discontinued work on the ptb promoter, in the interest of time. Friday, July 17 ● Plasmid N- and C-terminal protocols were accidentally swapped and were redone. ● Continued to refine the S. sediminis gene fragments and primers. ○ Need to manually reduce the GC content for ordering. Monday, July 20 ● Continued to work on the S. sediminis fragments and primers. Tuesday, July 21 ● Still working on the S. sediminis fragments and primers. ● According to Timpson et al., adh2 is an alcohol dehydrogenase in H. volcanii that shows high rates of butanol production. ○ Butanol production via reduction only occurs in acidic conditions. ○ The reverse oxidation reaction occurs in alkaline conditions. ○ Since H. volcanii is grown in alkaline conditions, oxidation is favored. ○ Considered growing H. volcanii and acidifying the environment. ● H. volcanii also has two aldehyde dehydrogenases (aldY3 and aldY5). ○ It’s unclear which results in more butanol production. ● Shifted the project to focus on developing two fusion genes consisting of adh2 and each of the aldehyde dehydrogenases. ● Started researching linker sequences to connect the two dehydrogenase genes. Wednesday, July 22 ● Finalized the S. sediminis fragments and primers. ● Made minimal media for growing H. volcanii anaerobically. ○ Will give several days for colonies to grow, before acidifying the environment. ● Reduced the number of rare codons in the S. sediminis gene. ● Contacted Rob Franks about gas chromatography usage. ○ He said it should be able to handle slightly acidic conditions. ○ Need to prepare standards before running samples. ● Looked up sequences of aldY3, aldY5, and adh2 on KEGG. Thursday, July 23 ● Prepared the S. sediminis fragments and primers for ordering. ● Started researching linker sequences to connect the two dehydrogenase genes. ○ Found a paper that describes characteristics of good linkers (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/) ● Professor Bernick urged us to find natural linkers rather than designing our own. Friday, July 24 ● ?????? Monday, July 27 ● Designed primers with Dominic’s help for amplifying aldY3 and adh2. ● Tried using Pfam to view the domains of fusion proteins to obtain natural linker sequences. Tuesday, July 28 ● Used InterPro to visualize the linker region in natural fusion genes. ● Met with Professor Bernick and established a long-term game plan: ○ Design primers for dehydrogenase genes such that they incorporate the linker region. ○ Use Gibson assembly to incorporate the dehydrogenase genes and linker into the pTA963 plasmid. ○ Transform the Gibson product into E. coli, and determine which colonies were successful in taking up the fusion gene by colony PCR. ○ Design sequencing primers and send product off for Sanger sequencing. ○ Use nested PCR on the colony PCR product. ● Dominic confirmed that he also has adh2 primers, so we only designed the aldY3 primers. ● Continued searching for linker sequences. Wednesday, July 29 ● Several linker regions were designed from scratch. Started to incorporate overlap with dehydrogenase genes. ● Other linkers were found to be conserved across several species. ● Finalized aldY3 primers for ordering. ● Settled on 4 linkers: 2 designed and 2 conserved. Thursday, July 30 ● Received the S. sediminis primers and diluted them to 100 uM. August 2015 Monday, August 3 ● Waiting for S. sediminis gene blocks. ● The anaerobic cultures haven’t grown much - pH still at 7.5. Tuesday, August 4 ● Received native gene primers. These primers were also diluted to 100 uM. ● Genomic DNA isolated from H. volcanii ○ Followed protocol in pg 70 of Halohandbook. Wednesday, August 5 ● Ran PCR on genomic DNA to isolate aldY5 (annealing temp 71℃). ○ Extension time should’ve been 1 min, but was only 30 sec. ● Ran a gel on the PCR product - mostly primers showed up with little to no PCR product. Thursday, August 6 ● Redid PCR of aldY5 and aldY3 using these conditions: ● aldY3 and aldY5 were successful, adh2 was not. ● Received the S. sediminis gene blocks. Friday, August 7 ● Ran PCR again on aldY5 and aldY3 using these conditions: ● Used a touchdown PCR on adh2 for more specific primer-binding (decrease annealing temperature by 0.5 ℃ each cycle). ● The conditions were successful Saturday, August 8 ● Re-ran PCR on adh2. ● Ran PCR on the 8 gene/linker fragments (71℃ annealing, 1 min extension, 2 min final hold) ○ aldy3BFw & aldy3Blink1Rev = A ○ aldy5BFw & aldy5Blink1Rev = B ○ aldy3BFw & aldy3Blink2Rev = C ○ aldy5BFw & aldy5Blink2Rev = D ○ aldy3BFw & aldy3Blink3Rev = E ○ aldy5BFw & aldy5Blink3Rev = F ○ aldy3BFw & aldy3Blink4Rev = G ○ aldy5BFw & aldy5Blink4Rev = H Monday, August 10 ● Unable to proceed due to a lack of supplies. Tuesday, August 11 ● Still unable to proceed further due to a lack of supplies. ● Anaerobic cultures appear to be growing. ● Started to prepare minimal HEPES media rather than Tris (pH 6.55). Wednesday, August 12 ● Continuing to make minimal media. ○ Considering eliminating potassium phosphate to avoid precipitate formation. ● Professor Bernick suggested micro-aerobic cultures, in which a small amount of oxygen is injected into the culture vessel. ○ May help solve the redox imbalance from the lack of oxygen as a terminal electron acceptor. ● Assembled S. sediminis gene fragments. ● Ran a gel with aldY5/adh2 and S. sediminis fragments. Thursday, August 13 ● The S. sediminis fragments were re-assembled (increased annealing temperature to 67℃). ● Gene/linker sequences also ran again. ● Key: ○ Lane1: aldy5BFw & aldy5Blink4Rev = H ○ Lane2: adh2Dlink1Fw & adh2DRev = I ○ Lane3: adh2Dlink2Fw & adh2DRev = J ○ Lane4: adh2Dlink3Fw & adh2DRev = K ○ Lane5: adh2Dlink4Fw & adh2DRev = L ● PCR conditions: Friday, August 14 ● Assembled the aldY and adh2 linker fragments. ○ aldy3BFw & aldy3Blink1Rev & adh2Dlink1Fw & adh2DRev = F1 ○ aldy5BFw & aldy5Blink1Rev & adh2Dlink1Fw & adh2DRev = F2 ○ aldy3BFw & aldy3Blink2Rev & adh2Dlink2Fw & adh2DRev = F3 ○ aldy5BFw & aldy5Blink2Rev & adh2Dlink2Fw & adh2DRev = F4 ○ aldy3BFw & aldy3Blink3Rev & adh2Dlink3Fw & adh2DRev = F5 ○ aldy5BFw & aldy5Blink3Rev & adh2Dlink3Fw & adh2DRev = F6 ○ aldy3BFw & aldy3Blink4Rev & adh2Dlink4Fw & adh2DRev = F7 ○ aldy5BFw & aldy5Blink4Rev & adh2Dlink4Fw & adh2DRev = F8 ● PCR conditions for fragment assembly: ● Samples were purified using a DNA cleanup kit, but purification was unsuccessful (see last lane). Sunday, August 16 ● Attempted to determine why the cleanup kit didn’t work. ○ Tested the reagents in the kit. ○ Still unsuccessful. ● Decided to not assemble fragments and then use Gibson assembly, but to go straight into Gibson assembly and see if the fragments assemble properly before annealing to the plasmid. Monday, August 17 ● Continued to work on cleanup kit. ○ Sample G1 was successfully cleaned up. ● Samples B, E, F, L need to be re-cleaned, from the kit mishap. ○ Despite cleanup, these samples had a low 260/230 value. ● Unsure whether to use N-terminal or C-terminal plasmid for our Gibson reaction. Tuesday, August 18 ● Confirmed that the N-terminal plasmid is required for Gibson of native fusion genes. ● Considered the idea of testing cultures for fermentation products such as lactic acid. ○ Rob Franks suggested using HPLC, since lactic acid won’t run in the column. ○ HPLC also somewhat non-specific. ● Ran Gibson assembly on A + I (F1) and B + I (F2) Wednesday, August 19 ● Gibson assembly failed due to a bad plasmid. Need to redo plasmid before attempting Gibson assembly again. ● Conditions used in generating the C-terminal and N-terminal plasmid: ● Started to figure out the details surrounding micro-aerobic cultures and how much oxygen needs to be added. Thursday, August 20 ● Recreated samples B, E, L, F. ● Recreated both N- and C-terminal plasmid. ● Results of plasmid (left) and samples B, E, L, F (right): ● Ran Gibson assembly on both S. sediminis samples and A + I again. ● Inoculated 3 micro-anaerobic cultures containing the following types of media: ○ HEPES buffer ○ DMSO ○ Unbuffered ● Transformed the A + I Gibson product into E. coli using heat shock and plated on ampicillin plates. Friday, August 21 ● Plates did not show growth, so transformation was unsuccessful. ● Planned to re-plate and/or possibly rerun Gibson assembly using a different ratio of plasmid to fragment. Monday, August 24 ● Created both buffered and unbuffered spheroplast solutions. ● Considered switching to transformation by electroporation rather than heat shock. Tuesday, August 25 ● Transformed E. coli with F3/G3 and C-terminal S. sediminis Gibson products using electroporation. Wednesday, August 26 ● Ran Gibson assembly on all remaining gene/linker fragments. Thursday, August 27 ● Ran colony PCR on G1 sample. ● Prepared samples and standards for gas chromatography of micro-aerobic cultures (using ethyl acetate as solvent). ○ 1% 1 mL butanol standard ○ 1% 1 mL lactic acid standard ○ 1 mL ethyl acetate blank ○ 10% 1 mL samples of each culture (Unbuffered, HEPES, DMSO, and aerobic) ● Generate more I fragment to rerun Gibson assembly on G2 (B + I). ● Ran colony PCR on G3 (C + J). Monday, August 31 ● Transformed and inoculated G4 and G5 into E. coli. ● Prepared 18% MGM to be used in establishing a new aerobic culture. September 2015 Tuesday, September 1 ● Ran colony PCR on G1, G4, and G5. ● Ran Gibson assembly on G2. ● Inoculated new aerobic cultures. Once grown, these cultures will be switched to anaerobic/micro-aerobic environments.