Team:Exeter/lab book/1gp/wk12

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ExiGEM2012 Lab Book 1GP wk12


Protocols \| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase
9th - 13th July - 16th - 20th July - 23rd - 27th July - 30th July - 3rd August - 6th - 10th August - 13th - 17th August - 20th - 24th August - 27th - 31st August - 3rd - 7th September - 10th - 14th September - 17th - 21st September - 24th - 28th September
	Single Gene Plasmids and Enzyme Characterisation: 24th - 28th September 2012 Monday 24th September (9.00) • Adding cultures with pBAD(weak)+RBS-GFP, pLacI/Ara-1+RBS-GFP and pLacI/Ara-1+RBS-WclY-terminator into liquid medium and incubation overnight Uninduced and induced (L-arabinose for pBAD(weak) and IPTG for pLacI/Ara-1) tubes for each of the three constructs were added along with chloramphenicol (for pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP) and ampicillin (for pLacI/Ara-1+RBS-WclY-terminator) as the selection antibiotics. pLacI/Ara-1+RBS-WclY-terminator was used to determine basal levels of GFP fluorescence. Tuesday 25th September (9.00) • Measuring GFP fluorescence of pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP Same method as followed for protein expression on Thursday 20th September, but 6 conical flasks were used for testing of pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP as well as determining basal levels for pLacI/Ara-1+RBS-WclY-terminator. However, 750µL was only transferred to cuvettes for measuring OD600 at 2 and a half hours after putting all 6 conical flasks in the shaking incubator. The results for OD600 measured is as follows: o 1a = 0.134. \| o 1a = 0.922. o 1b = 0.162. \| o 1b = 1.368. o 2a = 0.149. \| o 2a = 0.965. o 2b = 0.144. \| o 2b = 0.958. o 3a = 0.155. \| o 3a = 1.041. o 3b = 0.137. \| o 3b = 1.009. At: 9:45 and 12:15 left to right respectively ------------------ 1 = pLacI/Ara-1+RBS-WclY-terminator 2 = pLacI/Ara-1+RBS-GFP 3 = pBAD(weak)+RBS-GFP ------------------ a = uninduced (- inducer) b = induced (+ inducer) ------------------ Since OD600 = approximately 1.000, GFP fluorescence was then measured by aliquoting 200µL of each cultured conical flask to respective wells on a 96-well plate and then measuring fluorescence with settings: 480nm excitation, 530nm emission. The following measurements for GFP fluorescence were obtained: o 1a = 41285A. o 1b = 42191A. o 2a = 45381A. o 2b = 45462A. o 3a = 45220A. o 3b = 43045A. Where 'A' is absorbance. There wasn't any conclusive evidence that GFP fluorescence was higher in the + induced GFP constructs than the - induced GFP constructs and GFP fluorescence remained similar to basal levels with - and + induced pLacI/Ara-1+RBS-WclY-terminator. Wednesday 26th September (9.00) •