In our project, one of the major goals is to reduce the uremic toxin, p-Cresol. Clostridium bacteria inside the gut will ferment excess tyrosine into p-Cresol. With the help of tyrosine ammonia-lyase (TAL) , we are able to convert this excess tyrosine into a harmless product, p-Coumaric acid instead of the toxic p-Cresol. As such, the concentration of p-Cresol can be reduced.
For our construct design, we focused on BioBrick BBa_I742148 from the 2007 iGEM Edinburgh team in the iGEM BioBrick library. This enzyme has been shown to have the highest Km value compared to other TAL enzyme.
We hypothesized that the native ribosome binding site (NRBS) from Saccharothrix espanaensis will not have a high translation efficiency in E. coli Nissle, so we changed the NRBS in front of TAL into a strong ribosome binding site B0034. Also, we noticed that there was no spacer sequence between NRBS and the TAL coding sequence(CDS) in BBa_I742148.
According to research, the length of the spacer sequence between RBS and CDS strongly affects translation efficiency. We decided to follow the standard iGEM BioBrick assembly rule to add 6 bp of scar sequence between RBS and CDS. The schematic below depicts how we improved the BioBrick.
We wanted to prove that TAL expression was improved with the change of RBS (BBa_I742146 into B0034). First, RT-PCR experiment was used to confirm that the constructed TAL Biobrick is being transcribed. As seen in Fig.2, cDNA for both bacteria carrying TAL constructs are being detected by PCR, thus confirming that the TAL genes is actually being transcribed in E. coli Nissle.
|1||PCR positive control||8||BBa_K2997009 cDNA||3+4|
|2||BBa_K2997009 cDNA||1+2||9||BBa_K2997010 cDNA||3+4|
|3||BBa_K2997009 RNA||1+2||10||BBa_K2997009 RNA||3+4|
|4||BBa_K2997009 plasmid||1+2||11||BBa_K2997010 RNA||3+4|
|5||BBa_K2997010 cDNA||1’+2||12||BBa_K2997009 plasmid||3+4|
|6||BBa_K2997010 RNA||1’+2||13||BBa_K2997010 plasmid||3+4|
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. 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.
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