July
July 16
Public
Preparation of three strains with plasmid pCS-pgm-galu(kan), pSA-ARO10-adh6(cm) and pZE-UGT85A1
July 17
Public
Design of primers (pSA/pCS/pZE-KpnI-R、pZE-Xbal-F、pSA/pCS-BamHI-F、OsSJt1-Kpn1-F、OsSJt1-BamHI-R、pgm-galu-KpnI-F、pgm-galu-BamHI-R、pgm-galu-XbaI-R、aro10-adh6-KpnI-F、aro10-adh6-XbaI-R、aro10-adh6-BamHI-R)
Construction of original engineered strains using DH5α chassis.
July 18 - 20
Public
Amplification of engineered strains by transferring into liquid medium.
Verification of plasmid vectors and target genes using PCR and sequencing. pZE, pCS, UGT sequencing failed.
July 21
Gene Knockout Part
Inoculation of pCas and pTarget strains.
July 22
Gene Knockout Part
Primers for knocking out (p1, p2, p3, p4 of pykA, pykF, pheA) designed.
Preservation, plasmid extraction for pCas and pTarget strains.
July 23
Gene Knockout Part
Transformation of pCas and pTarget plasmid into chassis, which was later transferred on solid medium for overnight incubation.
Plasmid extraction of pCas strains, concentration evaluated.
July 24
Gene Knockout Part
Colonies of pCas and pTarget strains are selected and transferred into liquid medium for amplification.
Fermentation Part
Inoculation of pSA, pCS, pZE plasmid strains into liquid medium, incubate overnight.
July 25
Gene Knockout Part
Preservation, plasmid extraction, sequencing of pTarget strain. (No signals upon sequencing)
pCas induction succeeded. Preparation of pCas plasmid competent strains. Preservation under -80℃.
July 26
Fermentation Part
Inoculation of pSA, pCS, pZE strains on solid medium.
Gene Knockout Part
Amplification of pTarget strains in liquid medium.
July 28
Fermentation & Gene Knockout Part
PCR verification and sequencing of pSA, pCS, pZE, and pTarget strains. pTarget induced successfully. pSA and pCS also showed positive results.
July 30
Fermentation Part
Colonies of pheA knockout and pSA strains are selected for amplication. Later plasmid extraction was made.
Gene Knockout Part
PCR mutation, enzyme digestion for pTarget of pykA and pykF. Transformation into chassis which was later transferred on solid medium.
July 31
Gene Knockout & Fermentation Part
Preparation and transformation of pheA donor.
Amplification of pSA, pZE, pCS, pTarget, pheA, pykA, pykF strains.
August
August 1
Fermentation Part
Preservation, plasmid extraction, sequencing of engineered strains.
Construction of original engineered strains succeeded according to PCR verification results. Transferred for fermentation.
August 2
Gene Knockout Part
Preparation and transformation of pykA donor.
Preparation and transformation of pTarget of pheA and pykF.
August 3
Copy Number Optimization Part
Recombination of plasmids using vectors and function genes.
Gene Knockout Part
Transformation of pykA pTarget into pCas.
Colonies of pheA and pykF are selected and transferred into liquid medium for amplification.
August 4
Gene Knockout Part
Preservation, plasmid extraction, sequencing of pheA, pykF strains.
August 5
Copy Number Optimization Part
Plasmid recombination failed. pZE was changed to pET. Extract original engineered plasmids(pSA, pET, pCS)
Re-PCR verification using newly designed primers.August 6
Gene Knockout Part
Re-preparation of pTarget for pykF and pheA.
Amplification of engineered plasmid strains.
August 7
Gene Knockout Part
Preparation of pTarget plasmids.
Attempts for recombination of plasmids.
Transformation of pykA donor.
August 8
Copy Number Optimization Part
Verification of recombinant plasmid strains. Positive results except pCS. Positive plasmids are extracted and sent for sequencing.
August 9
Gene Knockout Part
Sequencing for pheA knockout strain succeeded. Transfer it into liquid medium for amplification and plasmid elimination.
Copy Number Optimization Part
PCR verification for recombinant plasmids.
August 10
Fermentation Part
Fermentation succeeded. Samples prepared for HPLC analysis.
Gene Knockout Part
Extraction and sequencing of pykF pTarget.
Preservation of pheA knockout strain after liquid cultivation.
August 11
Fermentation Part
HPLC analysis for fermentation samples.
Gene Knockout Part
Preparation of pCas competent strain using DH5α chassis.
Transferring pykF pTarget strain into liquid medium.
August 12
Fermentation Part
Fermentation showed desirable yields of rhodioloside in fermentation, but the standard sample had problems.
Copy Number Optimization Part
1 of 6 recombinant plasmids failed upon PCR verification.
Gene Knockout Part
Plasmid extraction and sequencing for 5 recombinant plasmids and pykF pTarget plasmid.
PCR verification for pheA knockout strain checking its status of knockout, pTarget and pCas induction. Electrophoresis show that pheA has been successfully knocked out.
August 13
Copy Number Optimization Part
Recombinant plasmids sequencing all showed negative results.
Construction of recombinant plasmids.
Gene Knockout Part
Preservation for pheA knockout strain.
Attempt to knockout pykA gene using donor and pTarget prepared.
August 14
Gene Knockout Part
PCR verification again for pheA knockout strain.
Transferring pheA knockout strain on solid and liquid medium for further investigation, according to the electrophoresis result.
August 15
Gene Knockout Part
Preservation and sequencing for pheA knockout strain.
PCR verification for pykA knockout strain. All pykA knockout samples turned out failed according to electrophoresis results.
Preparation of pykF pTarget. Transformation after preparation.
August 16
Copy Number Optimization Part
PCR verification for recombinant plasmid strains. Strains showing positive results are transferred to liquid medium.
Gene Knockout Part
Amplification for pykF pTarget strain in liquid medium. Strain preserved and sent for sequencing after amplification.
Verification for pheA knockout strain after further cultivation. All pheA knockout samples turned out failed according to electrophoresis results.
August 17
Copy Number Optimization Part
All recombinant plasmid strains in liquid medium cultivated successfully, plasmids extracted and sent for sequencing.
Gene Knockout Part
Microscopy for pheA knockout strain. E.coli taking the major part with a little contamination by mixed bacteria could be observed.
pykF pTarget plasmid sequencing failed. No signal.
August 18
Fusion Expression Part
New primers for knockout result sequencing and fusion expression plasmid synthesis designed.
Cyclization PCR for pykF pTarget plasmid construction.
Copy Number Optimization Part
Original engineered plasmids(pSA, pET, pCS with their original functional genes) went through PCR verification.
August 19
Copy Number Optimization Part
Extraction of pCS, pET, pSA plasmids, later went through concentration, enzyme digestion and separation to obtain vectors and functional genes for plasmid recombination.
Gene Knockout Part
PCR verification for pykF pTarget strain. Succeeded according to gel electrophoresis. Later colonies are selected for amplification in liquid medium.
PCR verification of pheA knockout strain and pkyA knockout strain using newly designed improved primers. pheA knockout strain showed negative results(all 3k bp bands instead of 2k) and pkyA knockout strain showed doubtful results. pykA knockout strain was left for further investigation.
August 20
Gene Knockout Part
Plasmid of pykF pTarget are extracted, sent for sequencing, and its concentration was measured.
Construction of pheA knockout and pykA knockout strain by transformation.
Copy Number Optimization Part
Verification of six vectors and genes for recombinant plasmids. Construction of recombinant plasmids after verification succeeded. Recombinant plasmids are Transformation into chassis.
Fusion Expression Part
Construction and purification of fusion genes adh6-linker-ARO10. (only first 2 linkers)
August 21
Fusion Expression Part
Construction of fusion genes adh6-ARO10. Another 4 linkers constructed and purified.
2 fusion genes made on Aug 20 collected, enzyme-processed and linked to pSA vector to form fusion expression plasmid. Plasmids with adh6-ARO10 fusion gene are Transformation into chassis.
Copy Number Optimization Part
PCR Verification for recombinant plasmid strains. Failed.
New plasmid vector strains(pACYCduet1, pCDFDuet1, PETDUET1, pRS-hemF) arrived. Incubate overnight.
Gene Knockout Part
Colonies of pykF pTarget selected for amplification in liquid medium.
August 22
Fusion Expression Part
The rest of fusion genes (linkers 3 to 6) enzyme-processed and linked to pSA vector to form fusion expression plasmids. Transformation into chassis.
Copy Number Optimization Part
PCR verification for recombinant plasmid strains. Strains with positive results transferred into liquid medium for amplification.
Plasmid extraction for new plasmid vector strains.
Primer design for new primers for verification, rPCR and Gibson Assembly® method.
Gene Knockout Part
pykF pTarget sequencing failed. SgRNA part of its gene sequence was wrong.
August 23
Gene Knockout Part
Preparation of pykF pTarget plasmid using new primers and construction of pykf pTarget strain.
Preparation of pheA donor.
Copy Number Optimization Part
Primer design for new primers for verification, rPCR and Gibson Assembly® method. Ready to send for synthesis.
Positive recombinant plasmid strains transferred into solid medium to form colonies.
Fusion Expression Part
PCR verification for fusion plasmid strains linker 1 and 2. Failed.
Liposome Vesicle Part
Launched. Confirmation of protocols. Attempts for preparation of vesicles.
RNA Thermometer Part
Launched. Confirmation of protocols. Strains with report gene EGFP ready.
August 24
Gene Knockout Part
Preparation of pheA and pykA donor. Construction of knockout cells by transforming corresponding donor and pTarget into pCas competent cells.
Colonies of pykF Target transferred into liquid medium for amplification.
Copy Number Optimization Part
PCR verification for positive recombinant strains in August 23. Failed.
Attempt of Gibson Assembly® method by building pETDuet1-ARO10-adh6 recombinant plasmid. Transferred into DH5α chassis.
Fusion Expression Part
PCR verification for fusion plasmid strains linker 1 to 6. Positive strains transferred into liquid medium for amplification.
Liposome Vesicle Part
Vesicles with desirable diameter could be observed. Later investigation in progress.
RNA Thermometer Part
Construction of report plasmid pET-EGFP. Transferred into DH5α chassis.
August 25
Gene Knockout Part
pykF pTarget plasmid extracted and sent for sequencing.
Copy Number Optimization Part
Construction of recombinant plasmid strains (vectors pET, pSA, pCS).
Attempt of Gibson Assembly® method. Failed in rPCR of the vectors.
Fusion Expression Part
Positive strains in Aug 24 transferred into solid medium.
Liposome Vesicle Part
Preparation of vesicles using different lecithin-cholesterol ratios. 7:4 found to be the optimal ratio for now.
RNA Thermometer Part
pET-EGFP strain colonies formed. Ready for verification.
August 26
Gene Knockout Part
PCR verification for pykA and pheA knockout strain colonies. Strains with positive results transferred into liquid medium for amplification.
pykF pTarget sequencing failed.
Copy Number Optimization Part
PCR verification for recombinant plasmid strains (vectors pET, pSA, pCS). Positive strains transferred to liquid medium for amplification. Strains with doubtful results transferred to solid medium.
Gibson Assembly® for single gene(ARO10-adh6, UGT) on three vectors(pCDFDuet, pETDuet, pACYCduet). Six recombinant plasmids in total. Transformation into DH5α chassis and incubate overnight.
Fusion Expression Part
PCR verification for positive strains in Aug 25. Strains with positive results transferred into liquid medium for amplification.
August 27
Fermentation Part
Fermentation strains (strains with original engineered pSA, pET, pCS plasmids) transferred into solid medium for colonies to form.
Gene Knockout Part
pheA knockout strain transferred to solid medium.
Preparation of pykF pTarget plasmid. Transferred into chassis.
Copy Number Optimization Part
Preservation and plasmid extraction for remaining positive recombinant plasmid strains (pET-ARO10-adh6 and pSA-UGT). Plasmids sent for sequencing.
New plasmid strains ready for verification.
Fusion Expression Part
Positive adh6-ARO10 fusion gene strains transferred to solid medium for further verification.
RNA Thermometer Part
Colonies of EGFP report strains were selected and went through PCR verification. Colonies with positive results transferred into liquid medium.
August 28
Fermentation Part
Colonies inoculated into liquid medium. Incubate overnight.
Copy Number Optimization Part
PCR verification for new plasmid strains and the rest of recombinant plasmid strains with doubtful positive results. Strains with positive results transferred to liquid medium.
Re-construction of new plasmid strains(pETDuet, pACYCduet) which showed failed in last verification PCR.
Fusion Expression Part
PCR verification for positive adh6-ARO10 fusion gene strains. Strains with positive results transferred to liquid medium.
Gene Knockout Part
pykA knockout strain transferred to solid medium.
Colonies of pykF pTarget strain transferred into liquid medium for amplification.
RNA Thermometer Part
Plasmids of EGFP report strains extracted, sent for sequencing.
August 29
Fermentation Part
Strains in liquid medium transferred into flask for fermentation.
Copy Number Optimization Part
PCR verification for the rest of recombinant plasmid strains. Failed according to gel electrophoresis results.
Plasmid extraction and preservation for remaining recombinant plasmid strains which showed positive results in the recent verifications. Plasmids sent for sequencing.
Fusion Expression Part
PCR verification for the rest of adh6-ARO10 fusion gene strains. Failed according to gel electrophoresis results.
Plasmid extraction and preservation for remaining adh6-ARO10 fusion gene strains which showed positive results in the recent verifications. Plasmids sent for sequencing.
Gene Knockout Part
PCR verification for pheA knockout strains. Failed.
Plasmid extraction, and preservation for pykF pTarget strains. Plasmids sent for sequencing.
RNA Thermometer Part
Sequencing succeeded for EGFP plasmid.
Tentative design for RNA thermometer.
August 30
Fermentation Part
First and Second samplings.
Preparation of standard rhodioloside sample.
Copy Number Optimization Part
Sequencing failed. Re-inoculation of strains preserved.
Gene Knockout Part
pykF pTarget sequencing failed. Re-inoculation of strains preserved.
New strategies for gene knockout using other sgRNAs and CRISPR-transposon system. Design of new primers.
Fusion Expression Part
Sequencing failed. Re-inoculation of strains preserved.
August 31
Fermentation Part
Third sampling. Samples sent for HPLC analysis.
Copy Number Optimization Part
Strains inoculated yesterday sent for sequencing.
PCR verification for colonies of strains pCDFDuet-ARO10-adh6 and pCDFDuet-UGT. Colonies showing positive results were transferred into liquid medium for amplification.
Gene Knockout Part
PCR verification for pykA knockout strain. Some colonies have completely knocked out the pykA gene according to electrophoresis results. Transferred into liquid medium for amplification.
New pheA pTarget plasmids prepared and Transformation into chassis.
Fusion Expression Part
Strains inoculated yesterday sent for sequencing.
RNA Thermometer Part
RNA thermometer connected with vector pET-EGFP then Transformation into chassis.
September
September 1
Gene Knockout Part
Colonies of pheA pTarget selected and transferred into liquid medium for amplification.
PCR verification for the pykA knockout strain. All showed positive results. Strains were then transferred to another liquid medium for elimination of pTarget plasmid.
pykA knockout strain sent for sequencing.
RNA Thermometer Part
PCR verification for colonies of RNA thermometer strains constructed yesterday. Colonies with positive results transferred into liquid medium for amplification.
September 2
Fermentation Part
Reports of HPLC arrived. Further data collection in progress.
Gene Knockout Part
pykA knockout strain transferred to solid medium for further screening of pTarget-eliminated strains.
Extraction of pheA pTarget plasmid. Sent for sequencing.
Sequencing of pykF pTarget using bacteria solution failed. Its pTarget plasmid sent for sequencing instead.
Copy Number Optimization Part
Sequencing using bacteria solution failed. Revelant strains re-inoculated into solid medium.
Fusion Expression Part
Sequencing using bacteria solution failed. Revelant strains re-inoculated into solid medium.
RNA Thermometer Part
Extraction of pET-RNA thermometer-EGFP plasmid. Sent for sequencing.
Gene Integration Part
Launched. Inoculation of pDonor, pEffector and pCutamp plasmid strains into liquid medium.
September 3
Gene Knockout Part
Screening for pTarget-eliminated pykA knockout strain.
pheA pTarget and its donor Transformation into pCas strain to build pheA knockout strain.
Copy Number Optimization Part
Colonies inoculated yesterday were selected and went through verification PCR.
Fusion Expression Part
Preparation of adh6-ARO10 fusion gene (with linkers 1 to 6). Ready for connection with vector pET.
RNA Thermometer Part
Sequencing succeeded. Transformation of U6, U7 and U8 - pET plasmids into chassis to construct reporter strains.
Gene Integration Part
Extraction of pEffector, pDonor and pCutamp plasmids. Re-inoculation of pEffector and pCutamp strains due to too low concentrations.
September 4
Gene Knockout Part
Elimination of pTarget plasmid for pykA knockout strain seems failed. Re-inoculation into liquid medium with inducer.
Verification PCR for pykF pTarget. Mostly negative results.
Copy Number Optimization Part
Construction of plasmids pCDFDuet-ARO10-adh6, pACYCDuet-ARO10-adh6 and pETDuet-ARO10-adh6. Transformation into chassis.
Verification PCR for recombinant plasmid strains recently constructed. Mostly negative results.
Fusion Expression Part
Construction of pET-(adh6-ARO10 fusion gene) plasmids with linkers 1 to 6. Transformation into chassis.
RNA Thermometer Part
Report strains transferred into liquid medium. Inducer IPTG added.
Gene Integration Part
Extraction of pEffector and pCutamp plasmids.
September 5
Gene Knockout Part
Verification PCR for pheA knockout strain. Strains with positive results transferred into liquid medium.
Screening for pTarget-eliminated pykA knockout strain.
Copy Number Optimization Part
Verification PCR for recombinant plasmids(vectors pSA, pET and pCS) all failed.
Doubtful results shown during verification PCR for new vector strains(pETDuet, pCDFDuet, pETDuet). Strains left for further investigation.
Fusion Expression Part
Verification PCR for pET-(adh6-ARO10 fusion gene) plasmid strains inoculated yesterday. Strains with positive results transferred into liquid medium for amplification.
RNA Thermometer Part
Solution of EGFP report strains selected and checked for fluorescence under naked eye. No visible phenomenon.
September 6
Gene Knockout Part
Verification PCR for pheA knockout strains in the liquid medium. All positive results. Transferred into liquid medium with inducer for elimination of pTarget.
Screening for pTarget-eliminated pykA knockout strain.
Copy Number Optimization Part
Verification PCR for new vector strains(pACYCDuet, pCDFDuet, pETDuet). Failed.
Fusion Expression Part
Plasmids of fusion genes with positive results extracted and sent for sequencing.
Re-construction of fusion gene plasmids which failed in the last attempt. Transformation into chassis.
RNA Thermometer Part
Solution of EGFP report strains selected and checked for fluorescence after excitation by UV light. No visible phenomenon.
Gene Integration Part
Plasmids sent for sequencing.
Design of relevant primers to perform gene integration using CRISPR-transposon method.
September 7
Gene Knockout Part
Screening for pTarget-elimated pykA knockout and pheA knockout strains.
Construction of pykF pTarget plasmid using new primers. Transformation into chassis.
Copy Number Optimization Part
Design of new primers for Gibson Assembly® method.
Fusion Expression Part
Verification PCR for fusion gene plasmids constructed yesterday. Failed.
Gene Integration Part
Construction of pDonor plasmids with ARO10 and with UGT85A1 gene. Transformation into chassis.
September 8
Gene Knockout Part
Screening for pTarget-elimated pykA knockout and pheA knockout strains.
Verification PCR for pykF pTarget strains. All positive. Strains transferred into liquid medium for amplification.
Copy Number Optimization Part
Construction of recombinant plasmids using vectors pSA, pET and pCS. Ready for biotransformation.
Gene Integration Part
Verification PCR for constructed pDonor plasmids(both with ARO10 gene and with UGT gene). Doubtfuls results shown. Later check for actual structure of the plasmid.
September 9
Gene Knockout Part
Verification PCR for pTarget-eliminated pheA knockout strain. Strains transferred into liquid medium for amplification.
Screening for pTarget-eliminated pykA knockout strain.
Copy Number Optimization Part
Construction of recombinant plasmid strains by biotransformation.
Construction of plasmid pCDFDuet, pACYCDuet and pETDuet linked with UGT85A1 gene. Transformed into chassis.
Fusion Expression Part
Fusion gene plasmids sequencing successful.
RNA Thermometer Part
Primitive report strain(without RNA thermometer) inoculated into liquid medium. Inducer IPTG added.
September 10
Gene Knockout Part
Verification PCR for pTarget-eliminated pheA and pykA knockout strain. pTarget of pheA knockout strains all eliminated while pykA stayed the previous results.
pTarget-eliminated pheA knockout strain preserved and transferred into liquid medium for pCas plasmid elimination.
Continue pTarget elimination for pykA knockout strain.
Copy Number Optimization Part
Verification PCR for recombinant plasmid strains and new vector plasmids(pETDuet, pACYCDuet, pCDFDuet with gene UGT). Strains showing positive results transferred into liquid medium.
RNA Thermometer Part
Report strains showed green color under naked eye. Relevant strains preserved.
September 11
Gene Knockout Part
Screening for pTarget-eliminated pykA knockout strain and pCas-eliminated pheA knockout strain.
pykF pTarget sequencing succeeded. Ready for biotransformation for construction of its knockout strain.
Copy Number Optimization Part
Extraction of recombinant plasmids. Sent for sequencing.
Gene Integration Part
Preparation of pDonor plasmid using new primers. Transformed into chassis.
RNA Thermometer Part
Inoculation of report strains U6, U7, and U8 into liquid and solid medium.
September 12
Gene Knockout Part
Screening for pTarget-eliminated pykA knockout strain and pCas-eliminated pheA knockout strain.
Construction of pykF knockout strain using biotransformation of donor prepared and pTarget.
Copy Number Optimization Part
Sequencing of pACYCDuet-UGT and pETDuet-UGT plasmids successful. Ready for later assembly of ARO10-adh6 gene.
Sequencing of pCS-ARO10-adh6 failed.
Gene Integration Part
Verification PCR for pDonor strains(with ARO10 and with UGT gene). pDonor plasmids extracted and sent for sequencing.
RNA Thermometer Part
Verification PCR for report strains (with RNA thermometers). Strains with positive results transferred into liquid medium for amplification.
September 13
Gene Knockout Part
Verification PCR for pTarget-eliminated pykA knockout strain and pCas-eliminated pheA knockout strain. Gel electrophoresis result and screening result indicate that pCas plasmid of pheA knockout strain has been eliminated while pTarget of pykA knockout stayed doubuful results.
pheA strains showing positive results transferred into liquid medium without antibiotics for amplification. pykA knockout strains transferred into liquid medium with inducer and corresponding antibiotics to eliminate pTarget plasmid.
Gene Integration Part
Plasmid extraction for pDonor plasmids.
pEffector plasmids sequencing failed. Sent for sequencing again with PCR products.
RNA Thermometer Part
Culture medium of report strains with thermometer U6, U7, U8 transferred into well plates to observe luminescence of EGFP.
September 14
Gene Knockout Part
Preservation of plasmids-eliminated pheA knockout strain. Preparation of competent cells.
pykA knockout strain transferred into solid medium for later screening.
Verification PCR for pykF knockout strains. No evidence of gene knocked out. Continue cultivation to knock out the gene.
RNA Thermometer Part
Luminescence observation failed due to wrong experiment facilities.
September 15
Gene Knockout Part
Verification for pheA knockout competent cells prepared yesterday. Inoculation of pheA knockout strain in case that competent cells not qualified for further biotransformation.
Verification PCR for pykF knockout strain. Failed. Retry the biotransformation.
Screening for pTarget-eliminated pykA knockout strain.
Preparation of pCas strain using biotransformation.
Gene Integration Part
Biotransformation of pDonor-UGT and pDonor-ARO10 plasmid into chassis.
September 16
Gene Knockout Part
Verification of pheA knockout competent cells failed. Results indicating that cells were unable to absorb the plasmids. Colony of pheA knockout strain selected and inoculated into liquid medium.
Colony of pCas strain selected and inoculated into liquid medium.
Screening for pykA knockout strain showed that strains having pTarget eliminated appeared. Verification PCR for pykA knockout strain.
RNA Thermometer Part
Preparation of report strains U6, U7, U8 using biotransformation.
September 17
Fermentation Part
Construction of engineered strains (pETDuet-ugt + pSA-aro10-adh6, pacyc-ugt + pSA-aro10-adh6, pacyc-ugt + pet-adh6-aro10(linker 3), pacyc-ugt + pet-adh6-aro10(linker 4)) prepared for later fermentation.
Gene Knockout Part
Verification of pykF knockout strain. Failed. Biotransformation for construction of pykF knockout strain.
pTarget-eliminated pykA knockout strain transferred into solid medium for later screening for pCas elimination.
Preparation of pheA-knockout competent cells. Inoculation test for verification.
Copy Number Optimization Part
Construction of pACYCDuet-ARO10-adh6-UGT85A1 plasmid. Transformed into chassis.
Gene Integration Part
Verification PCR for pDonor strains to seek for successfully integrated gene. Failed.
September 18
Fermentation Part
pETDuet-ugt + pSA-aro10-adh6 plasmid strain failed to grow. Verification PCR for the rest of fermentation strains constructed yesterday. Strains with positive results selected and inoculated into liquid medium for amplification.
Gene Knockout Part
Verification for pheA-knockout competent cells successful.
Screening for pCas-eliminated pykA knockout strain.
Copy Number Optimization Part
Verification PCR for pACYCDuet-ARO10-adh6-UGT85A1 plasmid strain. Strains with positive results selected and transferred into liquid medium for amplification.
Gene Integration Part
Verification PCR for pDonor strains to seek for existence of relevant plasmids. Successful. Results indicating that the plasmids still exist but no integration happened.
September 19
Fermentation Part
Fermentation strains preserved from inoculum.
Gene Knockout Part
All pCas plasmids eliminated in pykA knockout strain. Colonies selected and transferred into liquid medium for amplification.
Verification PCR for pykF knockout strain. Failed.
Copy Number Optimization Part
pACYCDuet-ARO10-adh6-UGT85A1 plasmid extracted from inoculum and sent for sequencing.
September 20
Gene Knockout Part
Preparation of pykA knockout competent cells.
Gene Integration Part
Verification PCR for integration strains. Strains with positive results transferred into liquid medium for amplification. PCR products sent for sequencing.
September 21
Gene Knockout Part
Verifivation of pykA knockout competent cells failed as wrong antibiotics was used. Verification again. Preparation of new pykA knockout competent cells in case it fails.
BL21-pCas strain inoculated into liquid medium for preparation of pCas competent cells.
Copy Number Optimization Part
pACYCDuet-ARO10-adh6-UGT85A1 sent for sequencing using new primers.
RNA Thermometer Part
Report strains inoculated into solid medium for colonies.
Gene Integration Part
Strains with positive results in verification PCR and sequencing transferred into liquid medium without antibodies to eliminate the tool plasmids.
September 22
Gene Knockout Part
Verification PCR for pykF knockout strain which failed on last trial. Positive results revealed. Strains with positive results inoculated into liquid medium for amplification.
Verification of pykA knockout competent cells successful.
Preparation of pCas competent cells canceled as all the desired genes in the project have been knocked out.
Copy Number Optimization Part
Plasmid pACYCDuet-ARO10-adh6-UGT85A1 sequencing succeeded. Ready for biotransformation.
RNA Thermometer Part
Colonies of report strains selected and inoculated into liquid medium for amplification.
Gene Integration Part
Strains going through plasmid elimination inoculated into solid medium to form colonies.
September 23
Fermentation Part
Construction of fermentation strain pACYCDuet-ARO10-adh6-UGT85A1 by biotransformation.
Gene Knockout Part
Preservation of pykF knockout strain and inoculation into medium with inducer IPTG for pTarget plasmid elimination.
RNA Thermometer Part
Inoculation of report strains into 24-well plates.
Gene Integration Part
Screening of plasmid-eliminated strains.
September 24
Fermentation Part
Verification PCR for fermentation strain pACYCDuet-ARO10-adh6-UGT85A1. Succeeded. Colonies selected and inoculated into liquid medium for amplification.
Construction of pheA-knockout-pSA-pET and pykA-knockout-pSA-pET strain by biotransformation. (pSA meaning pSA vector and its correspond gene ARO10-adh6, same for pET, etc.)
Gene Knockout Part
Inoculation of pykF knockout strain into solid medium for later screening.
RNA Thermometer Part
Concentration and fluorescence measured for report strains with time as the variant.
September 25
Fermentation Part
Preservation of fermentation strain pACYCDuet-ARO10-adh6-UGT85A1.
Verification PCR for pheA-knockout-pSA-pET and pykA-knockout-pSA-pET strain. Succeeded. Transferred into liquid medium for amplification.
Gene Knockout Part
Screening for pTarget-eliminated pykF knockout strain.
RNA Thermometer Part
Concentration and fluorscence measured for report strains at 18h.
Extraction of new report plasmid pET-28a. Construction of U6-1, U6-2, U7, U8, U9 report plasmids using this new backbone. Construction of new report strains using biotransformation.
September 26
Gene Knockout Part
Screening for pTarget-eliminated pykF knockout strain succeeded. Strains selected and inoculated into liquid medium for amplification.
September 27
Gene Knockout Part
pTarget-eliminated pykF knockout strain inoculated on solid medium to form colonies. Screening for pTarget-eliminated pykF knockout strain.
RNA Thermometer Part
pET28a plasmid sent for sequencing.
September 28
Gene knockout Part
pTarget-eliminated pykF knockout strains with positive results transferred into liquid medium for amplification.
RNA Thermometer Part
pET28a plasmids sequencing succeeded.
Verification for new report strains. Strains with positive results transferred into liquid medium.
September 29
Fermentation Part
Inoculation of fermentation strains on solid medium to form colonies.
Gene Knockout Part
Preservation of final pykF knockout strain.