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Part:BBa_M36154:Experience

Designed by: Monica Chin   Group: Stanford BIOE44 - S11   (2014-10-23)


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Stanford location of BBa_M36154 MUTANT

      • has ~400 bp deletion

Barcode #: 0133007587

Physical location: A1 of BioE.44 F2014 box

Plasmid name: MCAP nifA Sensor_mutant

Antibiotic resistance: ampicillin

DNA 2.0 Gene #: 19561

Organism: E.coli

Sensor

DNA 2.0 was unable to synthesize our construct. Moving forward, some adjustments are warranted to heighten the chances of synthesizing our DNA construct. The mutant that DNA 2.0 was able to synthesize contained a 356 base pair deletion in the nifA transcription factor sequence, implying that something in this sequence is producing problems during synthesis. One modification is to change the NifRLA and NifBQ promoters to constitutive promoters. This would eliminate a source of uncertainty in the device design since it is unclear if these promoters function properly in E.coli. It may also be the case that the nifA transcription factor is toxic to the cell. This is entirely possible seeing as how one of the main novelties of this proposed project was to determine if the nifA transcription factor could even be actualized in an E.coli system. There is data to suggest that the nifA sequence from certain organisms is inexpressible in E.coli, however, no studies thus far have shown that the Azotobacter vinelandii sequence is inexpressible. Before concluding that the Azotobacter vinelandii nifA sequence is inexpressible in E.coli, some additional analysis of the sequence itself should be done. The GeneDesigner software used for proofreading our construct may not have identified problematic repeat sequences. One should consider using more high-powered proofreading technology to confirm that there are no errors in the sequence itself before continuing with experimentation.

While we were unable to test our experimental design, experimentation with our positive and negative controls in varying nitrogen concentrations shows that E. coli cells are able to grow in environments that are nitrogen enriched. Furthermore, expression of the green fluorescent protein (GFP) in the p82 plasmid is uninhibited by the presence of excess nitrogen in growth media. The positive control had higher levels of fluorescence than the negative control; however, some experimentation needs to be done to minimize background fluorescence of the negative control. These findings show that certain modifications are necessary to controls prior to experimenting with the device. Furthermore, some changes in device design and proofreading techniques could be done to increase the likelihood of synthesizing the construct.

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