ENGINEERING

Laccase

  • Design: This part consists of a pH-inducible promoter, secretory signal peptide, laccase enzyme-coding sequence, a Tandem GS linker, and a reporter gene. The pH-inducible promoter, Asr (which stands for acid shock RNA), gets activated in the pH range of 3.5-5.5. With the help of the secretory signal peptide, the laccase enzyme is secreted extracellularly. In order to confirm the secretion of the laccase enzyme at the laboratory level, we added the reporter gene (CFP) in fusion with the laccase gene using a 13 Tandem GS linker.
  • Build: The gene circuit design has been constructed using SnapGene.
  • Test: ABTS assay is a common method to characterize the activity of the enzyme laccase. In this assay, ABTS, which is a colorless substrate, is oxidized by laccase, leading to the formation of a yellow-green product that can be measured at 420 nm. The absorbance value of the product correlates to the activity of laccase. Laccase was added to ABTS, and at regular time intervals, the absorbance was measured, and thereby the activity of Laccase was characterized
  • Learn: We initially faced issues with the design of the gene circuit, like the distance between RBS and the gene of interest, so that gene expression occurs properly. We had discussions with our alumni, experts, and figured out the issue, and modified the circuit.

Cry2Aa

  • Design: This part consists of a pH-inducible promoter, a Cry2Aa-coding sequence, a Tandem GS linker, and a reporter gene. The pH-inducible promoter, Alx (which stands for Alkaline Xylanase), gets activated in the pH range above 7. In order to confirm the secretion of the Cry2Aa protein at the laboratory level, we added the reporter gene (YFP) in fusion with the laccase gene using a 13 Tandem GS linker.
  • Build: The gene circuit design has been constructed using SnapGene
  • Test: In this experiment, the plasmid pUC19 carrying the gene of interest- CRY2Aa, was chemically synthesized and introduced into competent E. coli bacteria through transformation. Once the transformation procedure was completed, the E.coli was cultured, and the transformed colonies were distinguished from the non-transformants by inoculating them on an agar plate infused with ampicillin. After completion of screening, pUC19 was isolated from E. coli, and the extracted plasmid carrying Cry2AA was then run and visualized on the 0.8% agarose gel with a marker. This confirms the presence of the plasmid in E. coli and proves its integrity.
  • Learn:We faced a few issues in the transformation experiment; we had to change our protocol according to the experiment and got the results.

pAsr

  • Design: This part consists of a pH-inducible promoter and a reporter gene. The pH-inducible promoter, Asr (which stands for Acid Shock RNA), gets activated in the pH range below 7. In order to check the promoter working, this construct has been designed.
  • Build: The gene circuit design has been constructed using SnapGene.
  • Test:
    • Asr promoter (Pasr) is reported to be induced under acidic conditions.
    • It can be used as a reporter when the medium turns acidic. We thus measure the fluorescence intensity over a short period of time.
    • We first incubated the bacteria to the log phase (about 2 hours) in Luria-Bertani (LB) medium.
    • We then centrifuged the broth and resuspended the pellet using M9 medium with different pH values (pH 4, 4.25, 4.5, 4.75, 5, 5.5, 6, and 7; the pH value was adjusted with 1M HCl).
    • We then incubated it in the 96 well plates and measured its fluorescence intensity (absorbance: 485 nm, excitation: 535 nm) every 3 minutes for 30 minutes.
    • The difference in fluorescence intensity can be observed within 30 minutes.
    pasr

pAlx

  • Design: This part consists of a pH-inducible promoter and a reporter gene. The pH-inducible promoter, Alx (which stands for Alkaline Xylanase), gets activated in the pH range above 7. In order to check the promoter working, this construct has been designed.
  • Build: The gene circuit design has been constructed using SnapGene.
  • Test: With this construct, we showed that the alx promoter and riboswitch are able to regulate mNeonGreen expression, when pH is shifted to pH 8 and above. To do so, we let bacteria grow and then inoculated fresh media with adjusted pH (7, 8, and 8.5) to an OD600 of 0.2. After 20 and 40 min, 1 ml of each culture was taken and diluted to the lowest OD600 of the three samples, and then fluorescence and OD600 were measured with the plate reader (n = 3).
    palx