Part:BBa_K3040005

pFadBA promoter with consensus sequence regulating downstream RFP

Description

It is pointed out by the previous iGEM team, UPF CRG Barcelona_2018 iGEM, that the native promoter pFadBA (BBa_K817002) 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 by change the consensus sequence of -10 and -35 region.
According to the previous research, it suggests that this modification might improve the downstream protein expression.

Mechanism

Some study shows that in E. coli, there is sequence called consensus sequence which is universal and changes the expression level of downstream protein. A consensus sequence is existing in -10 and -35 region, so it is relative with gene expression.

Figure 1. This picture shows the consensus sequence in E coli and some mutant sequence of this region.

The expression level can be altered if the sequence is modified. The patterns of the data points indicate which promoter clones contained mutant in either the -35 or the -10 region with a consensus sequence.

Figure 2. This picture shows the activity of those sequence.

Method

1. Clone this promoter into DH5a
2. Detection fluorescence level after adding different concentration of fatty acid.
3. Analysis the data

Result

We detect the fluorescence by the mechanism. The following is the picture.

Figure 3. Detecting of the fluorescence level.

The data below shows that pfadBA-NTHU promoter has 2.1 folds increase in expression over native pFadBA as the concentration of fatty acid rises after 4 hours. In conclusion, this promoter has a higher fluorescence level in the higher fatty acid concentration environment.

Figure 4. 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).

This data shows the beginning fluorescence level of pFadBA_NTHU is much lower than pFadBA. Somehow, we successfully decrease the initial fluorescence by this modification and make an increasing rate can be more visible after inducing the downstream.

Figure 5. Detecting of the fluorescence level before adding fatty acid to induce E. coli.

In conclusion, we improve the fold change of pFadBA promoter. Compare with the data from iGEM12_NTU-Taida, the promoter (BBa_K817002) only rises for about 1.5 fold change after inducing. We modified the -10 and -35 region on FadBA with consensus sequence and successfully increase the fold change if we treat them with 7.5mM concentration of fatty acid. With higher concentration, get more downstream expression.

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.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 755
  Illegal AgeI site found at 867
  
• 1000
  COMPATIBLE WITH RFC[1000]