We were able to successfully clone our composite parts into DH5alpha E. coli cells, and Western Blots showed that the proteins were synthesized correctly. The bands were in the appropriate locations for the size of protein we were expecting.
This is an image of a Western Blot which shows that the mNG1-10 fragment was synthesized correctly.
This is an image of a Western Blot which shows that the mNG11 fragment was synthesized correctly.
Then, when the two populations of cells were cultured together, so that the medium was saturated with mNG11 and the other cell population contained mNG1-10 at high levels, lysis via sonicator showed that the split fluorophore came together to emit fluorescence at higher than background level.
In subsequent studies, there was a second positive result, in that lysing the cells with an antibiotic instead of sonication also generated an increase in fluorescence over time, as compared to the initial value. This suggests that the system works under the intended conditions. The fragments of mNeonGreen come together at a detectable rate after cell lysis.
The above graph demonstrates that 100 µl of Ampcillin (to a final concentration of 10 mg/mL) lyses the cells which retain the larger component of mNeonGreen (BBa_K2844009) at a rate sufficient for the first 10 strands of the beta barrel to join with the 11th strand (BBa_K2844006).
In terms of difficulties, we were unable to submit DNA samples of our basic parts because the cloning step failed. Essentially we were unable to successfully isolate plasmids that contained the coding sequences for the parts we were interested in submitting. We were able to submit DNA samples of our composite parts, but the plan for submission had to be modified at the last minutes. Moreover, our original intentions for the carrying out part improvement experiments failed as well. We wanted to compare and contrast the relative properties of the fluorescent proteins mOrange and mOrange2, but could not successfully clone them into pSB1C3 either, and so had to abandon that component of our plan. In addition, we struggled to clone mNeonGreen into the backbone pHISTEV to generate an overexpression plasmid.
We had great difficulty cloning our biofilm parts into pSB1C3 and transforming cells with them for this component of our project. In fact, the only successful transformation carried out was of the alginate binding protein fused to mCherry. This transformation did however produce a pale purple protein which, when purified, had the appropriate His-Tag suggesting that the protein synthesized was our target molecule, and not the Red Fluorescent Protein within the pSB1C3 backbone it was meant to have displaced.
This photo is of a column containing the fused alginate binding protein and mCherry, purified by means of a His-tag