Part:BBa_K4407016
vgb promoter with another FNR binding site inserted, microaerobic
This part contains vgb promoter (BBa_K561001) and an inserted FNR (fumarate-nitrate-regulator) binding site. vgb promoter is a microaerobic induced promoter from Vitreoscilla hemoglobin gene with an initial FNR (fumarate-nitrate-regulator) binding site in its sequence. In this part, we inserted another FNR binding site, of which the sequence was found on a research paper [1] as TTGATnnnnATCAA, upstream of the native binding site of the vgb promoter (BBa_K561001), in order to enhance the regulatory effect of FNR on vgb promoter. The regulatory effect on this part by oxygen concentration was more sensitive than that on vgb promoter (BBa_K561001) alone.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Results
1.Plasmid construction and transformation
We constructed a plasmid PMTL-FNRBS-Pvgb-bs2 to express the flavin mononucleotide based fluorescent protein Bs2 under the control of this promoter. Bs2 was provided by Dr. Zhengming Zhu’s research group. To construct the plasmid, we used the pMTL-Pvgb-bs2 plasmid constructed by team NJTech_China as template. Site-directed mutagenesis was carried out to obtain certain megaprimers by PCR technique using well designed primer, then another PCR technique was performed on pMTL-Pvgb-bs2 with megaprimers to obtain PMTL-FNRBS-Pvgb-bs2 recombined plasmid. Plasmids obtained were transformed into E. coli DH5α competent cells and cultured positive colonies for extraction of plasmid, which was then sent for sequence analysis, and correct ones were transformed into E. coli CA434 for conjugation with Clostridium tyrobutyricum.
2.Sensitivity of the promoter under aerobic and microaerobic conditions
The fluorescence intensity of the expressed Bs2 in the recombinant bacteria was measured as a reference to determine whether this promoter was more sensitive in regulating the downstream gene expressions in different oxygen concentrations than BBa_K561001. After collation and analysis of the fluorescence intensity data, the figure was draw as below (Figure 1). Promoter vgb is a hypoxia-induced promoter, and it’s expected that the higher the oxygen concentration is, the stronger inhibitory effect would be. Our modified strain bearing pMTL-FNRBS-vgb-bs2 plasmid showed higher inhibition than Pvgb-bs2 under aerobic condition, which indicated that our transformation was effective. Meanwhile, the modified strain FNRBS2-Pvgb-bs2 also showed inhibitory effect on the expression of downstream genes compared with Pvgb-bs2 under microaerobic condition.
Reference
[1]Chrystala Constantinidou, et al. A Reassessment of the FNR Regulon and Transcriptomic Analysis of the Effects of Nitrate, Nitrite, NarXL, and NarQP as Escherichia coli K12 Adapts from Aerobic to Anaerobic Growth. Journal of Biological Chemistry, Volume 281, Issue 8, 2006, Pages 4802-4815.
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