Difference between revisions of "Part:BBa K3040005"
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− | This data shows the beginning fluorescence level of pFadBA_NTHU is much lower than pFadBA. | + | This data shows the beginning fluorescence level of pFadBA_NTHU is much lower than pFadBA. Somehow, we successfully to decrease the initial fluorescence by this modification. |
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− | <div class="fig_title">Figure 5. Detecting of the fluorescence level.</div> | + | <div class="fig_title">Figure 5. Detecting of the fluorescence level before adding fatty acid to induce E. coli.</div> |
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− | + | In conclusion, 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. After we modified the -10 and -35 region with consensus sequence, the fold change can be successfully increase for more than 2 fold change. | |
Revision as of 00:47, 22 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 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.
![](https://static.igem.org/mediawiki/parts/0/05/T--NTHU_Taiwan--liya1.png)
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.
![](https://static.igem.org/mediawiki/parts/b/b0/T--NTHU_Taiwan--liya2.png)
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.
![](https://static.igem.org/mediawiki/parts/8/84/T--NTHU_Taiwan--liya3.png)
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 a higher fluorescence level in the higher fatty acid concentration environment.
![](https://static.igem.org/mediawiki/parts/1/17/T--NTHU_Taiwan--liya4.png)
This data shows the beginning fluorescence level of pFadBA_NTHU is much lower than pFadBA. Somehow, we successfully to decrease the initial fluorescence by this modification.
![](https://static.igem.org/mediawiki/parts/4/4a/T--NTHU_Taiwan--liya6.png)
In conclusion, 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. After we modified the -10 and -35 region with consensus sequence, the fold change can be successfully increase for more than 2 fold change.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 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]