July 21
Successfully extracted gltAB (pstar HS) from the genome of Pseudomonas aeruginosa, and it will be
amplified again. Enzymatic digestion and transformation coating are scheduled for tomorrow.
Extracted the genomes of both the first and second generations of Legionella pneumophila. PCR will
be
performed once the primers arrive tomorrow.
gltAB: Enzymatic digestion, ligation, and transformation coating have been performed. Tomorrow, the
growth status of the bacteria will be observed for verification, and they will be transferred to
test
tubes.
pgsBCA: After re-ligation and transformation yesterday, a total of 7 bacterial colonies have grown.
After
bacterial verification, there were no issues, and they have been transferred to culture tubes.
ureABC: Utilizing primers from the literature, ureABC has been successfully obtained from the
genomes of
both the first and second generations of Legionella pneumophila that were extracted earlier. It has
been
collected and will undergo enzymatic digestion tomorrow.
July 22
July 24
The pmv plasmid containing the racE gene (already extracted) has been purified. The racE gene has
been
connected using Gibson assembly and then subjected to transformation.
Similarly, ureABC and gltAB have been connected using redesigned primers via Gibson assembly and
subsequently transformed.
PMV-racE was extracted, and racE ureABC gltAB were obtained through Gibson primer PCR. Using Gibson
assembly, we constructed four plasmids: petlac-ureABC, pcs-gltAB, pcs-racE, and petlac-ureABC
connected
with T4. Transformation and coating processes were successfully completed.
Experimental Team Members: Yu Linwei, Liu Yazhao, Xie Wenjun.
July 25
July 26
Eight individual colonies from each of the ureABCx2, gltAB, and racE plates that were transformed
yesterday were selected.
After bacterial verification, the correct strains were transferred into test tubes. Plasmid
extraction
and subsequent uniform sequencing are planned.
Up to this point, all required plasmids have been successfully constructed (petlac-ureABC,
petlac-pgsBCA,
pcs-racE, pcs-gltAB, pcs-creJ, totaling five types, with a total of 12 plasmids). They have been
sent
for
sequencing by the company. If there are any sequencing errors, adjustments will be made accordingly.
For gltBD and glnA, which are the parts needed for CRISPR integration, genes have been extracted
from the
BW25113 genome. This yielded ygay up and ygaydown, two homologous arm segments, as well as the glnA
and
50xtrc promoter parts. However, obtaining gltBD has proven challenging, and we are currently in the
process
of adjusting the plan.
July 28
August 4
The glutamate synthesis pathway has been changed to GDH, and the desired integration fragment has
been
successfully constructed. It will be electroporated tomorrow.
All plasmids constructed previously have been sequenced correctly. Next steps involve changing the
required
promoters and separating and integrating different expression frames onto the designed plasmids.
The pcs-gltAB plasmid was extracted, and the expression frame and backbone were reverse cloned.
Simultaneously, the creJ expression frame was cloned out. After homologous recombination of these
two,
transformation coating was performed.
The integrated bacteria were selected and subjected to bacterial verification. Once confirmed to be
error-free, they were transferred to A+Sp medium.
August 10
August 17
Constructed PETlac-pgsBCAE, pCS-lac-racE, and pCSlac-gltAB plasmids.
Transformed these plasmids into our previously prepared host bacteria using electroporation and plated them for overnight cultivation.
Retrieved plated bacteria from the previous day and inoculated single colonies into test tubes for subsequent shaking flask fermentation.
Prepared substrate solutions required for fermentation.
August 18
August 19
Conducted fermentation verification for the first two modules and included a blank control group.
Completed the first batch of fermentation and collected samples for subsequent liquid-phase measurements.
Constructed all elements for sub-module validation.
August 22
August 28
Constructed PETduet-ureABC-lac-EFGD and, according to literature data, used BL21 as the vector to observe its inclusion bodies. We attempted to address the solubility issue of urease originating from Providencia species in the EcN vector.
Purified the fermented urease protein and characterized its solubility through SDS-PAGE electrophoresis.
August 31
September 4
Constructed pze-glnAP2-mcherry and attempted to induce glnAP2 expression under nitrogen-deficient conditions.
Created a random mutation library of glnAP2 promoter through error-prone PCR.
Designed experiments to integrate all three modules into one host bacterium.
Constructed pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE and pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmids.
September 10
September 12
Transformed pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE and pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmids into host bacteria and plated for antibiotic resistance screening.
Successfully cultured host bacteria containing pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE and pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmids.
Conducted fermentation validation of the entire synthetic pathway we designed.
September 14
September 15
Transformed host bacteria with the original plasmid pze-glnAP2-mcherry without a random mutation of the glnAP2 promoter.
Inoculated strains containing the pze-glnAP2-mcherry plasmid into test tubes for overnight cultivation.
September 16
September 17
Transferred strains containing the pze-glnAP2-mcherry plasmid into a 96-well plate with a series of ammonia concentration gradients.
After 24 hours of cultivation, measured the intensity of red fluorescent protein to characterize the sensitivity and detection limit of the glnAP2 promoter response to ammonia.
Transformed plasmid pze-glnAP2-random-mcherry with a random mutation of the glnAP2 promoter into host bacteria.
September 21
September 22
Inoculated 96 strains containing pze-glnAP2-random-mcherry plasmid into a 96-well plate for cultivation.
Transferred strains containing pze-glnAP2-random-mcherry plasmid into a 96-well plate with ammonia concentration gradients as per our experiment's settings.
After 24 hours of cultivation, measured the intensity of red fluorescent protein to conduct targeted screening of the random mutation.
September 23
October 1
Successfully obtained four random mutation promoters based on repeated experiments.
Designed a suicide system.
Began constructing pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmid.
October 2
October 6
Successfully constructed the pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmid.
Electroporated it into host bacteria for subsequent characterization.
Picked plated bacteria containing the pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmid and inoculated them into test tubes with different ammonia concentrations for overnight cultivation.
October 7
October 8
Measured the OD of bacterial cultures at each ammonia concentration to validate our designed suicide system.
Completed all experiments for the project.
October 10