Part:BBa_K4209004
cysP
Gene responsible for sulfate intake from Bacillus subtillis.
Introduction
This plasmid is used for sulfate intake in E.coli for zosteric acid production. Based on the literature, we found a way to produce zosteric acid (ZA) in E. coli BL21 DE3 strain by adding a sulfate group to coumaric acid. This is possible in cells thanks to two sets of genes: one essential for the transport of sulfate inside the cell and the other necessary for the catalysis of the phosphorylation. The first genes responsible for sulfate intake are cysP (from Bacillus subtillis) or cysPUWA (from E.coli). cysPUWA encodes an ATP-dependent sulfate transporter that generates a flow of sulfate ions towards the inside of the cell, while cysP encodes a proton-sulfate cotransporter that imports sulfate into the cell. TAL gene encodes for a tyrosine amino lyase (from Flavobacterium johnsoniae), which transforms L-tyrosine in p-coumaric acid. The p-coumaric acid will then be transformed in ZA thanks to the sulfotransferase encoded by SULT1A1.
Design
Ultimately, we designed our pathway to be split onto two separate vectors with one ‘transport’ plasmid, bearing the genes responsible for the intake of sulphate, and one ‘catalytic’ plasmid, bearing the genes responsible for the synthesis of ZA. In this page, we only discuss about the ‘transport’ plasmid. We obtained the genes for our transport vector, cysDNC, cysQ, cysPUWA and cysP, by PCR-amplifying them all from the genome of E. coli but for the latter, which we got from the genome of Bacillus subtilis. The ‘transport’ genes were to be cloned into the pCola_Duet vector.
Results
We successfully PCR-amplified all the genes and linear backbone fragments required to perform our cloning. Then we cloned the genes into their respective backbone through Gibson assembly and transformed the cloned plasmid into E. coli DH5α. By performing colony PCR and subsequent sequencing on the resulting transformants, we also were able to confirm the successful cloning of all of our plasmids.
The newly-built plasmids were then co-transformed into E. coli BL21 (DE3), a strain more adapted for protein production. Indeed, we wished to generate four distinct strains each containing different combination of two plasmids, one bearing sulphate uptake genes and one bearing the the catalytic enzymes. Unfortunately, we were not able to co-transform E. coli BL21 (DE3) containing pCola_Duet_P_DNCQ and pET_17b_Tal_SULT1A1. We, however, successfully obtained the three following co-transformant that we analyzed further for ZA production.
Check out our results part (https://2022.igem.wiki/unilausanne/results) from the wiki to have further information and see if we could produce zosteric acid.
Design Notes
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 62
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 62
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 62
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 62
Illegal NgoMIV site found at 538
Illegal NgoMIV site found at 656 - 1000COMPATIBLE WITH RFC[1000]
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