Part:BBa_K1163103
pAceB-LacI-LVA-Term
iGEM Evry 2013
Description:
This part is composed of the following elements:
- pAceB promoter region
- LacI-LVA
- Terminator
The AceB gene encodes the malate synthase A enzyme, which is involved in carbon source management. Its promoter region has been extracted from the genome of Escherichia coli. We found after genomic research that the gene is under the regulation of fur, thus meaning it is sensitive to iron concentration variations.
FUR (Ferric Uptake Regulation) is a transcriptional repressor of genes involved in iron homeostasis. In the presence of iron, FUR binds iron and dimerizes. This modification of conformation allows the linkage to a Fur Binding Site and inhibits the mRNA transcription of the downstream gene.
This 300 bp long promoter sequence contains a RBS and a FUR binding site, although it has not been clearly possible to identify them.
Why would you use this part?
This part is useful for iron sensing purposes in Escherichia coli. The pAceB promoter represses the downstream gene.
Characterization:
We characterized its inhibition ability by cloning a superfolder GFP downstream at a iron concentration range from 10-7 to 10-4 mol.L-1.
Additionally, we characterized our inverter by cotransforming with BBa_J04450 and measured the expression of RFP at a iron concentration range from 10-7 to 10-4 mol.L-1.
Compatibility
Chassis: Device has been shown to work in BBa_V1004
Plasmids: Device has been shown to work on pSB1A3
Devices: Device has been shown to work with BBa_K1163102, BBa_K1163103
Data:
Figure 1 Growth of E. coli strains containing either the A the pAceB-GFP iron sensor or the B pAceB-lacI+PL_lacO-RFP Fur inverter with the iron supplementations of either 0.1, 1, 10, 100 uM.
Fig 2 GFP expression of the pAceB-GFP Biobrick (BBa_K1163102) is repressed at higher iron concentrations. This construct thus functions an an iron-responsive biosensor to repress expression of the reporter gene GFP.
Fig 3 RFP expression of E. coli transformed with plLacO-RFP (Biobrick BBa_J04450) in presence (plates 1 and 2) and absence (plate 3) of IPTG. Adding IPTG increases RFP expression, confirming that Biobrick BBa_J04450 enables LacI-mediated RFP repression.
Fig 4 RFP expression as a function of iron concentration for E. coli expressing the Fur inverter. Cells were co-transformed with constructs bearing the pAceB-LacI and plLacO-RFP genetic elements. Together, these elements enable gene expression to be activated by elevated iron levels.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 277
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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