Part:BBa_K4632012:Design
nirB +dapA - Nutritionally deficient Biosafety device based on diaminopimelic acid (DAP)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 142
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Figure 1. Diagram of biological safety device circuit design
The initially designed experiment verification involved adding restriction enzyme sites at both ends of the pnirB, and then connecting the pnirB promoter to the pet-28b plasmid's EGFP front through enzyme digestion and ligation. The plan was to detect differences in promoter expression strength between aerobic and anaerobic environments by measuring fluorescence intensity.
However, during the actual experiments, multiple attempts failed to successfully construct the target plasmid. It was suspected that the pnirB promoter fragment might be too short (78 bp after adding the ribosome binding site, RBS), leading to a relatively low success rate of enzyme digestion and ligation. Subsequently, attempts to construct the plasmid using ΩPCR were unsuccessful. Finally, the design was modified to first connect the pnirB to the expression fragment and then construct the plasmid through enzyme digestion and ligation.
The original plan was to detect differences in promoter expression strength between aerobic and anaerobic environments by measuring fluorescence intensity. However, after further literature review, it was discovered that the previous oversight was the impact of anaerobic conditions on the ability of the fluorescent protein to emit fluorescence. As a result, EGFP was eventually replaced with the Gm resistance gene, and verification of pnirB was done through gradient concentration.
Source
nirB: https://parts.igem.org/Part:BBa_K4632008 dapA:https://parts.igem.org/Part:BBa_K4632005
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.
Dehio C, Meyer M. Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli[J]. J BACTERIOL, 1997,179(2):538-540.