Team:Pasteur Paris/Interlab study
The purpose of the Interlay Study is to compare the efficiency of three different promoters. In order to do this, we had to assemble the GFP gene (BBa_I13504) with each of the promoters in pSB1C3 and then transform it into DH5-ɑ cells. Two negative controls were performed: Bacteria transformed with I13504 in pSB1C3 and and mock-transformed WT bacteria. We observed them under UV light.
Building of the different constructions
- Each plasmid containing a promoter (BBa_J23101, BBa_J23106, BBa_J23117) was digested using Spe I and Pst I restriction enzymes.
- The GFP (BBa_KI13504) was digested using Pst I and Xba I restriction enzymes.
- Each promoter was ligated with the GFP and transformed in DH5-ɑ cells
- a quantitative PCR (qPCR) to determine the number of plasmids containing the GFP present in each strain.
- a fluorescence test to determine the GFP expression in each strain
→ First, We have performed an efficacy assay so as to elucidate the settings and the amount of template required for our qPCR. In order to do this, we amplified the TyrA gene, a single copy gene in E. coli in genomic DNA (gDNA)(WT strain), and GFP gene in plasmid DNA (pDNA) (GFP in pSB1C3 strain) at various concentrations (dilution series of 1/10). The results show that the qPCR for TyrA was efficient: ~1ng/µL is the amount of template to use.
→ Afterwards, We performed a parallel qPCR of our templates in order to amplify in one 96-well plate the correct amount of gDNA for each strain, and on another plate the amount of pDNA (GFP+) corresponding to each strain. A ratio of pDNA (GFP+) / gDNA was obtained: it shows the number of pDNA (GFP+) copies per biological clone and strain.
Results:
pDNA / gDNA Standard deviation PA 1.96 0.24 PB 2.62 0.83 PC 2.85 1.58
→ Finally, We analyzed the specific fluorescence of each strain and biological clone as a function of its growth since the latest iGEM data suggests that the GFP is expressed differently depending on the growth curve.
Knowing that the fluorescence depends on cell growth, we measured the fluorescence (508 nm) and the OD(600 nm) simultaneously thanks a TECAN robotic station equipped with a spectrophotometer.
Results:
Fluorescence Standard deviation PA 30794.41 670.92 PB 18780.67 772.80 PC 871.42 80.77 GFP 2234.05 85.26 PB 392.75 15.39
→ Thus, qPCR ratio values were used to normalize the data obtained from fluorescence and bacterial growth analyses, in order to examine each promoter's strength. In fact, the fluorescence obtained on each biological clone over time is divided by its qPCR ratio, in order to set the specific fluorescence value to a single pDNA (GFP+) devise. Therefore, by analyzing the differential of expression between strains and their biological clones, we determined which promoter triggered a higher GFP expression.
Results:
Technical replicates of the biological replicate PA:
Technical replicates of the biological replicate PB:
Technical replicates of the biological replicate PC:
Average for each biological replicates of the construct PA, PB ans PC and Controls:
Conclusion
We see that the promoter A (BBa_J23101) is the strongest of the three promoter that we had to test. The promoter B (BBa_J23106) also allows an improvement of the GFP expression. Unlike the two other promoter, the promoter C (BBa_J23117) doesn't improve the GFP expression, the results being the same as the controls.
After transformation of our Bacteria E. coli DH5-ɑ with each of our construct, we observed the cultures under UV light.
Promoter A(J23101) + GFP Promoter B(J23106) + GFP Promoter C(J23117) + GFP
WT (Non transformed cells) GFP(I13405)
For each clone, we made several analyses:
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