Part:BBa_K3040006
pFadBA_NTHU
It is pointed out by the previous iGEM team, UPF CRG Barcelona_2018 iGEM, that the native promoter pFadBA has great leakage and is a weak promoter that isn’t ideal enough for our system. Therefore, to enhance the sensitivity and reduce the leakage of the promoter pFadBA for making it more suitable for our system, we modified the native promoter pFadBA with a mutant in its consensus sequence of -10 and -35 region. According to the previous research [1], there is improvement in expression for pFadBA with consensus sequence.
Furthermore, the improved in expression portion enhance greatly if there’s a point of mutant in consensus sequence. Therefore, we designed our promoter pFadBA with a mutant in its consensus sequence of -10 and -35 region, to promote its strength.
Result
Though both pFadBA_NTHU (BBa_K3040005) and pFadBA_NTHU (BBa_K3040006) mutant have 1 to 2 fold increase in expression over native pFadBA as the concentration of fatty acid rises, pFadBA_NTHU has greater performance than pFadBA_NTHU mutant.
Thus, we could conclude that the modification of consensus sequence in -10 and -35 region could indeed improve the expression of the native promoter pFadBA, while the point mutation in advance had probably negative effects on its performance.
Figure 1. Relative protein expression of fatty acid promoter pfadBA-NTHU and pfadBA-NTHU mutant after 4 hours of induction under different fatty acid concentration (n=3).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 755
Illegal AgeI site found at 867 - 1000COMPATIBLE WITH RFC[1000]
Reference
JENSEN, Peter Ruhdal; HAMMER, Karin. The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl. Environ. Microbiol., 1998, 64.1: 82-87.
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