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 (first showing the 'pellet' samples, the second showing the 'supernatant' samples). 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)

• Running supernatant's of pBAD(large)+RBS-OmpA-SacB-terminator and TetR+RBS-OmpA-SacB-terminator (both induced and uninduced samples) on SDS-PAGE

Since we wanted to discover whether OmpA was exporting SacB out of the cell, supernatant's of the penultimate time frame (at 16.30 on Thursday 20th September) for just these two full constructs (1a, 1b and 2a, 2b respectively) were run again on SDS-PAGE.

The protocol was followed exactly, except:

o Tubes of 1a, 1b, 2a and 2b were briefly spun down before commencement with the procedure.

o 200µL of spun down supernatant was added to disposable columns and centrifuged at 6'000rpm for 5 minutes at 21°C and longer if necessary before all tubes reached the 50µL mark on the column scale.

o A stock of 2x LDS Sample Buffer and 2x Sample Reducing Agent was made by adding 125µL of 4x LDS Sample Buffer and 50µL of 10x Sample Reducing Agent to 75µL MilliQ H20. 50µL was then added to each of the four centrifuged supernatant samples (since ratio is 1:1).

o Samples were heated at 90°C instead of 70°C for 10 minutes.

o SDS-PAGE gel was run for 200V for an hour instead.

20µL samples were loaded in the order: Lane 1 = Protein ladder, Lane 2 = pBAD(large)+RBS-OmpA-SacB-terminator - inducer, Lane 3 = pBAD(large)+RBS-OmpA-SacB-terminator + inducer, Lane 4 = TetR+RBS-OmpA-SacB-terminator - inducer and Lane 5 = TetR+RBS-OmpA-SacB-terminator + inducer.

As can be seen, no protein of the correct molecular weight (SacB = 17,863Da) was observed.