Part:BBa_K1804002
pNirB-Invasin
In this construct, the invasin gene from Yersina is placed under the expression of the anaerobic-sensitive promoter pnirB from Escherichia coli.
Usage and Biology
In the facultative anaerobe Escherichia coli K-12, the transcription factor Fumarate and Nitrate reductase Regulatory (FNR) protein is encoded by the fnr gene (Spiro & Guest, 1987). FNR activates various fnr-dependent promoters in the anaerobic pathway of E. coli (Spiro & Guest, 1987), such as the nirB promoter (pnirB), which drives the expression of NADH-dependent nitrite reductase (Jayaraman et al., 1987). pnirB has partial activity when induced under anaerobic conditions and optimal activity in the presence of nitrite in addition to anaerobic growth conditions (Griffiths & Cole, 1987; Jayaraman et al., 1987). pnirB is a switch that can induce the expression of target genes under anaerobic conditions. As the non-pathogenic E. coli K-12 strain BL21 [http://www.ncbi.nlm.nih.gov/nuccore/CP010816.1] (Life Technologies) that we used expresses the fnr gene endogenously, pnirB can be regulated by FNR protein in this system.
Part Submitted
We have sequenced the part we submitted, and found that it contains the nirB promoter and the B0032 ribosome binding site, but we are unable to confirm if the full invasin sequence is present (the sequence readout was incomplete). The gel electrophoresis image of the colony PCR (done to confirm part size) is available on the Design page. The BioBricks Prefix and Suffix are present.
Results: Invasion Assay
We induced the expression of invasin by growing B21 carrying pSB1C3-pnirB-invasin under anaerobic conditions. After 2 and 6 hours, the bacteria were allowed to invade a monolayer of HepG2 mammalian cells in an invasion assay, where results are represented in colony-forming units (CFU) of bacteria successful in invading mammalian cells. Image is available as Figure 7 from: http://2015.igem.org/Team:SPSingapore/Anaerobic_Promoter
Our results show that BL21, and BL21 with pSB1C3-nirB-invasin grown under aerobic conditions have low invasion ability. BL21 has low invasion ability under both aerobic and anaerobic conditions . However, BL21 with pSB1C3-pnirB-invasin exhibits a 25-fold increase in invasion compared to the control grown at aerobic conditions, and this increase is statistically significant (p-value < 0.05) (Figure 11). It is notable that after 6 hours of growth in anaerobic conditions, the pnirB-invasin construct can no longer confer an increased invasive phenotype, which may be due to the high fitness cost of producing the transgenic invasin protein.
References
Griffiths, L., & Cole, J. A. (1987). Lack of redox control of the anaerobically-induced nirB+ gene of Escherichia coli K-12. Archives of microbiology, 147(4), 364-369.
Jayaraman, P. S., Peakman, T. C., Busby, S. J. W., Quincey, R. V., & Cole, J. A. (1987). Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite. Journal of molecular biology, 196(4), 781-788.
Spiro, S., & Guest, J. R. (1987). Regulation and over-expression of the fnr gene of Escherichia coli. Journal of general microbiology, 133(12), 3279-3288.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal NotI site found at 7 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
- 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal XbaI site found at 16 - 1000COMPATIBLE WITH RFC[1000]
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