Looking back on the last three weeks it felt like being a protein factory! Week 3 started very well with 2 new mutants and both of them were verified by sequencing, so I started overproducing InhA_WT and my 4 mutants in Week 4 and 5!
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| Part of my usual Day: Make Broth, Over-express InhA, Clean up! |
On a usual day, I would start setting up the over-expression first thing in the morning; whilst my transformed E.coli BL-21 were growing to the right density to be introduced, I would lyse the cell pellet from the previous day using a French press, so we could start the purification. That would keep me busy the whole day! Multi-tasking and keeping track of everything is really the key skill to have, filling time gaps with making more broth for the next day, checking OD of the 1L cultures, equilibration of the purification column, lysing more cells, cleaning up, preparing samples for SDS-Page gels, introducing the cultures, centrifugation with a temperamental Beckmann Avanti which is probably older than me and doesn't like to open the door once in a while...
...in theory, getting InhA should have been nice and easy with the His-Tag
and a GE His-Trap Nickel column...but...reality always looks a bit
different. First, I had no protein coming off the column, then too much causing precipitation with attempts to recover being unsuccessful. Now the fraction are pure, but can't quite concentrate them to a desired concentration of at least 20 mg/ml for the spectroscopy.
Sooo.... I kept over-expressing and over-expressing and over-expressing...guess it must have been about 50 I made in total in these two weeks, whilst Pauls's PhD Kirsty aka "the Protein Queen" kept purifying and purifying and purifying....I am very, very grateful for all her help!
Tomorrow (week 6), I will meet up with Dr Neil Hunt from our Physics department and his summer student to learn how to do FTIR (Fourier Transform Infrared) spectra of InhA and my mutants!
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| Checking the purity of my fractions |
Sooo.... I kept over-expressing and over-expressing and over-expressing...guess it must have been about 50 I made in total in these two weeks, whilst Pauls's PhD Kirsty aka "the Protein Queen" kept purifying and purifying and purifying....I am very, very grateful for all her help!
Tomorrow (week 6), I will meet up with Dr Neil Hunt from our Physics department and his summer student to learn how to do FTIR (Fourier Transform Infrared) spectra of InhA and my mutants!
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