Joanna Bird – Page 2

Bacterial Transformation, Mutagenesis and Protein Production

Joanna Bird / November 12, 2014November 16, 2014 / Protocols

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.

My M.Sc. in a Paragraph

Joanna Bird / November 3, 2014November 16, 2014 / General

My M.Sc. by research focuses on peptide-guided drug delivery, working on the conjugation of chemotherapeutics to neurotensin for tissue targeted drug delivery. As of October 2014, I am using techniques such as bacterial transformation, protein expression and purification, PCR, mutagenesis, SDS-PAGE and DNA electrophoresis regularly. My M.Sc. will expand in the coming months into tissue culture, a technique that I already have experience with. I have previously worked on projects that employed immunochemical staining and subsequent quantification. In particular, VEGF expression in breast cancer cell lines in relation to angiogenesis and the effect of anti-cancer drug resistance on this.

An Introduction to a Year in the Life

Joanna Bird / October 30, 2014November 16, 2014 / General

As of today, I’m a whole month into my MSc – my journey into becoming a better researcher, a better student, my initial introduction to true caffeine addiction (whatever you want to call it). I’d be lying if I said it has been easy so far, but easy does not get you anywhere in life so who wants that?

This month alone I have learnt several new practical techniques, some of which I have had to carry out for the first time with very little help, but with clear instructions. I’ve found that to be the most useful way, surprisingly. I’ve learnt what works for me without excessive external influence and I’ve learnt a great deal in the process.

I’ve optimised, calibrated, succeeded and failed. I’ve also been on BBC news, a glorious five seconds of fame – it was literally five seconds but still. I attended many seminars as an undergraduate, where the speaker would describe their own experiences as a postgraduate and the times they almost pulled their hair out trying to make their experiments work. I am not expecting this to be any different for me, nor would I want it to be. Because, it is not constant success that makes a good researcher. It’s the ability to not let failure alter your passion for the research, but let it simply educate you further.

I only aim to give all that I can, in the short time that I have to complete this M.Sc., in the hopes of being the best scientist I can be – all for my bloomin’ love of science. I hope that you enjoy my intermittent rambles, should you choose to follow me on this journey.