Part:BBa_K2616003
AgrC AgrA S. aureus detection system and RIP production under P2 promoter
In order to produce RIP only in presence of Staphylococcus aureus, we engineered a sensor device in our modified E. colicapable of detecting Staphylococcus aureus and producing RIP further to detection. This part contains under a constitutive promoter the sensor device of S. aureus agr operon, composed of agrA and agrC. If S. aureus approaches our system, AIP will be detected by transmembrane agrC,launching the phosphorylation of agrAand activating promotor P2. Our RIP sequence fused to a signal sequence for periplasmic export is placed after promotor P2 and will consequently be expressed only if S aureus approaches our biofilm.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 32
Illegal NheI site found at 55
Illegal NheI site found at 1533
Illegal NheI site found at 1556 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2769
- 1000COMPATIBLE WITH RFC[1000]
We gene synthesized our DNA commercially by Eurofins-Genomics. We received this genetic construct in three parts that we called Seq5 (1422 bp), Seq6 (960 bp) and Seq7 (762 bp) in the commercial plasmid pEX-A258 which we amplified in competent E. coli DH5α.
After bacterial culture and plasmid DNA extraction, we digested the commercial vector with XbaI and BamHI for Seq5, MscI, and SphI for Seq6, and HindIII and SpeI for Seq7. We extracted the insert from the gel and ligated by specific overlaps into linearized pBR322 for expression and into pSB1C3 for iGEM sample submission.
We had trouble to proceed with the ligation of the three inserts to linearized pBR322 and pSB1C3. We discussed with Takara Bio about our ligation issues, the GC percentage on our overlaps was too high to allow for a good ligation. Due to the lack of time, we were not able to redesign the overlaps for this construction.
| None |