UESTC-China

Early-stage Work

4.10 Team Members Confirmed

After the team competition and individual trials, 15 students were chosen as participants for iGEM 2023.

4.25 First Major Team Meeting (4.10-4.25)

Established the fundamental directions for air purification; selection of chassis organisms; modification of nicotine metabolic pathways; consideration of secondary purification; deliberation on using immobilized bacteria or immobilized enzymes for purification.

5.9 Second Major Team Meeting (4.26-5.9)

Finalized deodorization and second-hand smoke purification segments; identified basic substances for deodorization: butyric acid, indole, hydrogen sulfide, ammonia; confirmed E.coli as the chassis organism; determined the bacterial purification approach; further discussed the technical approach.

5.16 Third Major Team Meeting (5.10-5.16)

Discussed the feasibility and optimization of the technical approach.

5.24 Fourth Major Team Meeting (5.17-5.24)

Discussed specific strains of E.coli for construction; finalized plasmid vectors, screening genes, expression patterns, etc.; confirmed the gene knockout approach for indole and formaldehyde pathways.

5.25-7.9

Confirmed the expression vector sequences; refined the technical approach; outlined the preliminary experimental plan; awaiting gene synthesis; familiarized with laboratory equipment; undertaking end-of-term exams.

Formal Experiment Initiation

First Week 7.10-7.16

Plasmid Extraction

Cultivated bacteria from the gene synthesis company and extracted plasmids containing the target genes, preparing for vector construction.

Construction of Gene Expression Vectors

Attempted enzyme digestion and recovery of plasmids obtained from the plasmid extraction group, with a low recovery efficiency.

Gene Knockout

Conducted a pre-experiment to verify the effectiveness of the gene knockout primers; amplified the resistance segment using pKD4 as a template, but recovery was unsuccessful.

Weeks Two and Three (7.17-7.30)

Construction of Gene Expression Vectors

Completed the enzyme digestion and recovery of a majority of the synthesized plasmids.

Gene Knockout

  1. Successfully amplified the resistance fragment and recovered it.
  2. Employed a heat shock method to transform pKD46, resulting in theE.coli Top10/pKD46 strain.
  3. Prepared E.coli Top10/pKD46 electrocompetent cells.

Degradation Function Verification (Formaldehyde and Indole Degradation)

  1. Since several genes for formaldehyde and indole metabolic pathways are located on a single plasmid, assembly was unnecessary. The genes could be directly introduced into E.coli Top10 for degradation function verification.
  2. Attempted to extract bacterial proteins for SDS-PAGE to validate protein expression (unsuccessful).
  3. Validated the tolerance of E.coli Top10, which harbored the plasmid expressing isatin degradation enzymes, to varying concentrations of indole and isatin.

Week Four (7.31-8.6)

Construction of Gene Expression Vectors

  1. As certain genes related to nicotine, ammonia, and hydrogen sulfide couldn't be synthesized, we opted to replace the promoter and continue synthesis.
  2. Completed enzyme digestion and recovery for the remaining plasmids.
  3. Utilized Gibson assembly for the construction of tar and butyric acid metabolic pathways, and sent them for company sequencing, currently awaiting the sequencing results.

Gene Knockout

The knockout attempts from the previous week were verified via PCR and were deemed unsuccessful. Consequently, we are continuing the knockout process; however, false positives are occurring frequently, and even bacterial contamination has emerged. We are actively investigating the reasons for this and intend to refine the experimental methods before proceeding.

Degradation Function Verification (Formaldehyde and Indole Degradation)

  1. Validated the tolerance of E.coli expressing the formaldehyde degradation plasmid to varying concentrations of formaldehyde.
  2. Following the PI's advice, examined the growth curves of E.coli with the introduced formaldehyde degradation genes and indole degradation genes under conditions of 500μM formaldehyde concentration and 200mg/L indole, respectively.
  3. Conducted SDS-PAGE to validate the expression of enzymes related to formaldehyde degradation.
  4. Utilized a colorimetric method to establish a standard curve for formaldehyde concentration in the supernatant.

Weeks Five and Six (8.7-8.20)

Construction of Gene Expression Vectors

  1. Successfully synthesized the remaining genes for hydrogen sulfide and ammonia metabolic pathways, and initiated assembly and transformation;
  2. Due to recurrent assembly and transformation failures in the tar and butyric acid degradation pathways, we changed the promoter and continued attempts at assembly.

Gene Knockout

  1. We discovered that the purchased pKD4 plasmid differed from the standard, rendering the primers for amplifying the resistance segment ineffective. This necessitated re-synthesis, resulting in a significant time loss.
  2. Subsequently re-amplified the resistance segment, performed knockout, verified through PCR, and sent for company sequencing.

Degradation Function Verification (Formaldehyde and Indole Degradation)

Due to a mutation in the piGEM23_04 plasmid after its transfer into Top10, hindering normal expression, experiments and validation of the indole degradation pathway are currently being conducted in E.coli EPI400.

  1. Conducted SDS-PAGE to confirm the expression of formaldehyde degradation enzymes.
  2. Employed SDS-PAGE to verify the expression of the indole degradation pathway (fmo) and detect its product, indigo.
  3. Created standard curves for indole, isatin, and indigo for analysis.

Seventh Week (8.21-8.27)

Construction Of Gene Expression Vector

  1. Because some genes of nicotine cannot be synthesized, we replaced the promoter to continue the synthesis;
  2. Because the tar and butyric acid degradation pathway always fails to be assembled or transformed, assembly is still attempted after replacing the promoter.

Gene Knockout

Designed and synthesized the correct knockout primers based on the re-purchased pKD4 plasmid.

Degradation Function Verification

  1. Formation of inclusion bodies by formaldehyde pathway enzymes, hps, and phi.
  2. Verification of the formaldehyde pathway requires knockout of the frmA gene. Since the wild-type strain is capable of formaldehyde degradation, our approach involves introducing another pathway for degradation to avoid carbon dioxide production. Consequently, formaldehyde degradation function verification is temporarily suspended and will resume once the knockout is completed.
  3. Examined the production of indigo in the indole degradation pathway and plotted production curves.

Eighth Week (8.28-9.3)

Construction Of Gene Expression Vector

  1. After optimizing the promoter of the nicotine pathway, nicotine synthesis is continuing;
  2. Gibson assembled the tar and butyric acid metabolic pathways and sent them to the company for sequencing, and the sequencing results showed abnormalities.

Gene Knockout

  1. Utilized the newly synthesized knockout primers to PCR amplify the targeting segments and conducted recovery.
  2. Employed a heat shock method to transform pKD46, resulting in the E.coli Top10/pKD46 strain.
  3. Prepared E.coli Top10/pKD46 electrocompetent cells.

Degradation Function Verification

  1. Conducted qualitative assessment of indole degradation efficacy in the indole degradation pathway using Kovac's reagent.
  2. Quantitatively evaluated the effects of endogenous and exogenous indole degradation in the indole degradation pathway using Kovac's reagent.

Gene Expression Verification

  1. Culture bacteria expressing butyric acid and hydrogen sulfide degrading enzymes, and lyse bacteria to extract proteins.
  2. The protein expression and target bands were verified by polyacrylamide gel electrophoresis.

Ninth Week (9.4-9.10)

Construction Of Gene Expression Vector

  1. Successfully synthesized nicotine metabolic pathway genes, completed enzyme digestion and recovery;
  2. Re-assemble the two metabolic pathways of tar and butyric acid by Gibson, and send them to the company for sequencing, waiting for the sequencing results.

Gene Knockout

  1. Conducted electroporation of the targeting segments for the tnaA gene, resulting in the E.coli ΔtnaA::Kan/pKD46 strain.
  2. Selected transformants and used validation primers for PCR to confirm the successful knockout of tnaA.

Degradation Function Verification

  1. Considering the pale yellow color of isatin at low concentrations of isatin in water, switched to M9 medium for detecting isatin production.
  2. Compared the differences in indigo production in the indole degradation pathway under endogenous and exogenous indole conditions.

Gene Expression Verification

  1. Culture bacteria expressing butyric acid and hydrogen sulfide degrading enzymes, and lyse bacteria to extract proteins;
  2. Polyacrylamide gel electrophoresis was used to verify the protein expression and test the target bands;
  3. Failed to find the target band, continue the above experiment.

Tenth Week (9.11-9.17)

Construction Of Gene Expression Vector

  1. After the completion of nicotine enzyme cutting and recovery, assembly and conversion began;
  2. Re-assemble the two metabolic pathways of tar and butyric acid by Gibson, and send them to the company for sequencing, waiting for the sequencing results.

Gene Knockout

  1. Conducted electroporation of the targeting segments for the frmA gene, resulting in the E.coli △frmA::Kan/pKD46 strain.
  2. Selected transformants and used validation primers for PCR to confirm the successful knockout of frmA.
  3. Eliminated pKD46, resulting in the E.coli △frmA::Kan strain.

Degradation Function Verification

  1. Redrew the isatin detection standard curve in M9 medium.
  2. Tested isatin production in M9 medium and plotted the production curve.
  3. Compared the differences in indigo production in the indole degradation pathway under endogenous and exogenous indole conditions.

Gene Expression Verification

  1. Culture bacteria expressing butyric acid and hydrogen sulfide degrading enzymes, and lyse bacteria to extract proteins;
  2. Polyacrylamide gel electrophoresis was used to verify the protein expression and test the target bands;
  3. Failed to find the target band, continue the above experiment.

Eleventh Week (9.18-9.24)

Construction Of Gene Expression Vector

  1. Complete the nicotinase cutting and enzyme-linking work, and carry out the bacterial transformation work;
  2. Carry out Gibson assembly of nicotine metabolic pathway, and send it to the company for sequencing, waiting for the sequencing result;
  3. The two metabolic pathways of tar and butyric acid were re-assembled by Gibson and sent to the company for sequencing, and the sequencing results showed good results.

Gene Knockout

  1. Prepared E.coli △frmA::Kan in a heat-shock competent state, transferred pCP20 to E.coli △frmA::Kan, spread it on plates containing Amp and Kan, and incubated at 30°C overnight to eliminate the Kan resistance gene.
  2. Employed a heat shock method to transform pKD46 into E.coli △frmA.
  3. Prepared E.coli △frmA/pKD46 electrocompetent cells.

Degradation Function Verification

  1. Tested the production of isatin by the variant in M9 medium.
  2. Plotted the production curve of isatin for the variant and compared it with the wild type.

Gene Expression Verification

  1. Culture bacteria expressing butyric acid and hydrogen sulfide degrading enzymes, and lyse bacteria to extract proteins;
  2. Polyacrylamide gel electrophoresis was used to verify the protein expression and test the target bands;
  3. Failed to find the target band, continue the above experiment.

Immobilization of bacteria

Preliminary preparation of the light-reactive hydrogel.

Twelfth Week (9.25-10.1)

Construction of gene expression vector

  1. Carry out Gibson assembly of nicotine metabolic pathway, and send it to the company for sequencing, waiting for the sequencing result.

Gene Knockout

  1. Conducted electroporation of the targeting segments for the pgi gene, resulting in the E.coli △frmA△pgi::Kan/pKD46 strain.
  2. Selected transformants and utilized validation primers for PCR to confirm the successful knockout of pgi.

Degradation Function Verification

  1. Validated the isatin production capability of the piGEM23_04 variant.
  2. Plotted the production curve of isatin for the piGEM23_04 variant and compared it with the wild type.

Gene Expression Verification

  1. Culture bacteria expressing butyric acid and hydrogen sulfide degrading enzymes, and lyse bacteria to extract proteins;
  2. Polyacrylamide gel electrophoresis was used to verify the protein expression and test the target bands;
  3. Failed to find the target band, continue the above experiment.

Immobilization of bacteria

Preliminary preparation of the light-reactive hydrogel.

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