Team:Exeter/lab book/1gp/wk12

Operon Construction  |  Glycobase

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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.

Washing the stains of the two SDS-PAGE gels with MilliQ H20 revealed no distinct novel bands on either of the gels. It was thought that the concentration of proteins was too low for any such bands to be identified.

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

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1 = pLacI/Ara-1+RBS-WclY-terminator

2 = pLacI/Ara-1+RBS-GFP

3 = pBAD(weak)+RBS-GFP

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a = uninduced (- inducer)

b = induced (+ inducer)

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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)

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