Part:BBa_K4632008
Promoter(nirB-medium)
Description
nirB-medium is a synthetically engineered nirB promoter with the deletion of nitrite-responsive part ,thus, it is only able to induce heterologous genes in E.coli under low oxygen pressure or anaerobic conditions. (Rezaet al.,2014)
1. How does it work?
Wild type pnirB was isolated from Escherichia coli(E.coli), where it directs expression of an operon which includes the nitrite reductase gene nirB, and nirD, nirC and cysG. It is regulated both by nitrite and by changes in the oxygen tension of the environment, becoming active under anaerobic conditions. Response to anaerobiosis is mediated through the protein FNR encoded by the fnr gene. (Oxeret al.,1991)
2. What we have done?（SCAU-China 2023)
The initial experimental design involved connecting the promoter to the pet-28b plasmid's EGFP gene by adding enzyme-cutting ends to both ends of the pnirB fragment in nirB medium. The plan was to detect differences in promoter expression strength by measuring fluorescence intensity in aerobic and anaerobic environments (plasmid map detailed in Figure 1).
However, despite multiple repeated experiments, we were unable to successfully construct the target plasmid. It was speculated that the nirB promoter fragment, which was too short (78bp after adding rbs), resulted in a low success rate for enzyme-cutting connections.
Subsequently, we attempted to construct the plasmid using the Ω PCR method, but were still unsuccessful. Finally, we designed a new approach where we first connected the nirB fragment to the segment that needed to be expressed and then constructed the plasmid through enzyme-cutting connections.
During intermittent experiments, we further consulted the literature and discovered that an anaerobic environment could affect EGFP fluorescence expression, which was not conducive to measuring promoter expression strength.
Therefore, we replaced EGFP with the Gm resistance gene and characterized nirB through gradient concentrations.
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]
Construction and Characterization
We constructed pnirB-Gm by enzyme digestion and ligation (HindIII and PstI) onto the pBAD24M plasmid, which carries an ampicillin resistance gene. The transformed plasmid was selected using ampicillin resistance in E. coli DH5α. Finally, we characterized the expression strength of pnirB under aerobic and anaerobic conditions using gradient gentamicin resistance. Colony PCR gel electrophoresis results showed bands of the expected size (as shown in Figure 1).
Fig.1 Colony PCR of pBAD24M-pnirB-Gm resistant lane1-3: PCR of pBAD24M-pnirB-Gm resistant
The successfully transformed strains were streaked onto eight different plates with gentamicin concentrations of 0/0.5/1/2/5/10/15/30 μg/mL. These plates were incubated for 48 hours in two different environments: aerobic and anaerobic (placed inside an anaerobic chamber) conditions. (as shown in Figure 2).
Fig.2 Growth of E. coli DH5α containing anaerobic promoter and GM-resistant under different conditions.
Result: In aerobic conditions, bacterial growth was significantly inhibited at Gm 5 μg/mL. However, under anaerobic conditions, significant inhibition was observed at Gm 15 μg/mL.
References
Reza N, Akbari Eidgahi M R. Construction of a Synthetically Engineered nirB Promoter for Expression of Recombinant Protein in Escherichia coli[J]. JUNDISHAPUR J MICROB, 2014,7(6).
Oxer M D, Bentley C M, Doyle J G et al. High level heterologous expression in E. coli using the anaerobically-activated nirB promoter[J]. NUCLEIC ACIDS RES, 1991,19(11):2889-2892.
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