Part:BBa_K4209000:Design
tal-fjo, a gene encoding for a tyrosine amino lyase that converts Tyr to pHCA
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1303
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1303
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1303
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1303
Illegal AgeI site found at 925
Illegal AgeI site found at 1267
Illegal AgeI site found at 1357 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Design
For our project, we wished to produce ZA in large quantities using engineered bacterial cells as “cell factories” to assess its efficacy as an anti-foulant agent against quagga mussels. To be able to produce zosteric acid, we engineer plasmids containing SULT1A1 and TAL in different orders: one with SULT1A1 and then TAL and in the other direction. SULT1A1 and TAL were placed in a pET17b plasmid with an IPTG inducible promoter for protein expression in E.coli (see figure 2). The T7 promoter is activated once we add IPTG to the growth media, which switches on the expression of the genes.
SULT1A1 and TAL have been synthesized. We cloned the two plasmids individually in E. coli DH5α strain, then extracted them using a plasmid purification kit. We co-transformed E. coli BL21 DE3 cells with a plasmid construct containing SULT1A1 and TAL and a plasmid construct containing genes responsible for the sulfate intake to allow the production of ZA.
Successful co-transformants were isolated by plating cells on selective media supplemented with both the antibiotics kanamycin and ampicillin. The ZA produced was then released in the extracellular space and diluted in the media. We decided to use M9 as growing media for our transformed cells as it is a minimal media and contains low quantities of sugars and salts, making it easier to detect ZA. Detection of ZA was performed with high performance liquid chromatography (HPLC), as the sulfate group in the ZA made it undetectable using the GC-MS available in our department. Upon confirmation of the production of ZA, we have planned to concentrate and purify it using distillation. This would allow us to create either a liquid solution containing a high concentration of ZA or even a powder containing ZA and salts.