The first stage of my project required bacterial transformation for the production of two plasmids: pAG3 and pAG4 – aptly named after my project supervisor. The first is a Histidine-GFP-neurotensin containing plasmid and the latter lacks the neurotensin component as a result of mutagenesis. I used two strains for E.coli for this stage of the project – XL-1 blue and BL21 (DEM).
Site-directed mutagenesis to the pAG3 plasmid was conducted with an optimised PCR running programme and specially designed primers. The mutagenesis product was then digested by dpn1 to ensure that only the plasmid of interest was later transformed into XL-1 blue. XL-1 blue does not contain the T7 promoter region, meaning that this strain in particular is not able to produce protein from this plasmid, simply replicate the plasmid. The image below shows the product of this transformation. You can see the difference between the number of observable colonies between the transformation of pAG4 and pAG3 (scroll down for the other image)- this is typical for plasmids transformed as a product of mutagenesis.
As an experiment running alongside the mutagenesis of pAG3 to pAG4, I induced protein expression from the pAG3 plasmid in a series of steps:
- Transformation of pAG3 into BL21 E.coli cells (contains the T7 promoter – thus, produces protein)
- 5ml overnight culture of transformed cells from the previous step
- Expansion of culture in 200ml media
- Induction of protein expression with IPTG – incubated overnight
The first image above shows the colonies from transformation of pAG3 into BL21(DEM) (note how many more colonies are present compared to transformations of pAG4 into XL-1 blue) – this has a slight green colour due to the expression of the protein, which contains green fluorescent protein (GFP). The same applies to the second image of the overnight culture that had been IPTG induced the previous day – it appears rather green compared to the media it is grown in.
Next week: I will be purifying this protein with immobilised metal affinity chromatography and sending off pAG4 for sequencing.