Difference between revisions of "Part:BBa K4632008"

Latest revision as of 14:44, 12 October 2023

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

Construction and Characterization

pnirB promoter characterization

The pnirB-Gm fragment was cloned to pBAD24M through digestion and ligation. Three rounds of PCR amplification were employed to introduce the pnirB promoter and restriction sites in front of the gentamicin (Gm) resistance gene. Successful transformation of this construct into bacterial strains was confirmed through Gm resistance selection. The colonies that could grow on the Amp+Gm plate were further checked by colony PCR (Figure. 2). Subsequently, the expression strength of pnirB were assayed under aerobic or anaerobic conditions with a gradient concentration of Gm.

Figure 2. Colony PCR of pBAD24M-pnirB-Gm resistant

Lane1-3, PCR of pBAD24M-pnirB-Gm resistant

The successfully transformed strains were streaked onto eight different Gm concentration plates, ranging from 0/0.5/1/2/5/10/15/30 μg/mL. These plates were incubated for 48 hours under aerobic or anaerobic conditions (anaerobic conditions were achieved by placing the plates inside an anaerobic chamber).(Figure. 3)

Figure 3. Growth of E. coli Top 10 containing anaerobic promoter and Gm-resistant under different conditions

Under non-resistant and anaerobic conditions, bacterial growth is expected to be less favorable compared to aerobic conditions. As observed from the results, up to 2 μg/mL resistance concentration, there was no difference in bacterial growth between aerobic and anaerobic environments. Starting from 5 μg/mL, , only a few colonies were present in the initial streaked area under the aerobic environment. </p <p>In contrast,, bacterial growth was still extensive even at 10 μg/mL under anaerobic conditions. This suggests that pnirB is specifically expressed under anaerobic conditions. However, when the Gm increased to 15 μg/mL, strain could not grow well neither under aerobic nor anaerobic conditions.

In conclusion, the expression of pnirB under anaerobic condition is at least two times higher than the aerobic conditions. This could help us to construct the Biological Safety Device. See more detail in BBa_K4632012[1]

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.