Overview
In our project, the superfolder green fluorescent protein (sfGFP) allows better quantification of promoter strength and sensitivity.[1,2]. In the oxidative stress sensing system biobrick, we improved the biobrick BBa_K2610031 from the 2019 iGEM Leiden team by changing GFP (BBa_E0040)[2]. into sfGFP(BBa_I746916), which is hypothesized to have a higher expression level than GFP [Fig. 1]. We also added transcription activator SoxR to our biobrick for increased function of the oxidative stress sensing system[3].
Results
Disk assay
Disk assay was used to check the effect of each inducer. The concentration of the different inducers we used are listed below:
Inducer | Volume per disk |
---|---|
H2O2s (30%) | 10μl |
H2O2w (3%) | 10μl |
MD (menadione)(10mM) | 10μl |
DMSO (solvent for MD) | 10μl |
PQ (paraquat)(1mM) | 10μl |
MQ (solvent for PQ) | 10μl |
Then, we carried out SDS-PAGE to check the protein expression of TAL, both with TyrP and without TyrP. The expected protein size of TAL is 54 kDa and the expected protein size of TyrP is 43 kDa. As seen in the results below, however, there’s no distinguishable band around both sizes.
Finally, to confirm the protein activity of TAL and TyrP, we performed a functional test using n-octanol extraction method, which was previously proposed by iGEM_Uppsala 2013 and has been verified by HPLC[3]. The p-Coumaric acid concentration was measured through the absorbance value at 310 nm wavelength under Nanodrop UV-Vis wavelength.
We compared the TAL constructs containing the native and B0034 ribosome binding sites, (BBa_K2997009 and BBa_K2997010) to determine if p-Coumaric Acid production is improved by changing the ribosome binding sites. From the results seen in Fig. 4, BBa_K2997010 is able to produce a higher amount of p-Coumaric acid. Hence, we are able to prove that by changing the RBS (from Native to B0034), the conversion of tyrosine into p-Coumaric acid can increase by 1.73-fold. Therefore, we have shown that we have improved a previous BioBrick.
For more information, please visit our Results page.
References
- https://2007.igem.org/wiki/index.php/Edinburgh/Team
- Chen, H., Bjerknes, M., Kumar, R., & Jay, E. (1994). Determination of the optimal aligned spacing between the Shine – Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Research, 22(23), 4953–4957.
- https://2013.igem.org/Team:Uppsala