Difference between revisions of "Part:BBa K3040005"

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===Description===
 
===Description===

Revision as of 12:39, 20 October 2019

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 consensus sequence of -10 and -35 region. According to the previous research, there is improvement in expression for pFadBA with consensus sequence.

Mechanism

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

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


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

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.

Detecting of the fluorescence level.

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

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).

This data shows the beginning fluorescence level of pFadBA_NTHU is much lower than pFadBA.

Detecting of the fluorescence level.

Actually, we improve the fold change of pFadBA promoter. Compare with the data from iGEM12_NTU-Taida, the promoter (BBa_K817002) only rise for about 1.5 fold change. However, after we modified the -10 and -35 region with consensus sequence, the fold change can be successfully increase.



Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 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]


Functional Parameters