Team:Exeter/lab book/1gp/wk11

Operon Construction  |  Glycobase

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Tuesday 18th September (9.00)

• Mini-Prepping of TetR+RBS-WbnJ-terminator, constitutive promoter+RBS-WbnJ-terminator and pLacI/Ara-1+RBS-WfcA-terminator

• Gel Electrophoresis to check fragment sizes of TetR+RBS-WbnJ-terminator, constitutive promoter+RBS-WbnJ-terminator and pLacI/Ara-1+RBS-WfcA-terminator

• Transformation of pBAD(large)+RBS-OmpA-SacB-terminator, TetR+RBS-OmpA-SacB-terminator, TetR+RBS-Has-terminator (x 2), pLacI/Ara-1+RBS-WclY-terminator and TetR+RBS-WbnK-terminator

• Adding cultures with OmpA into liquid medium and incubation overnight

The following concentrations (in ng/µL) were obtained:

o TetR+RBS-WbnJ-terminator - 1. 375.3; 2. 377.9; 3. 509.3; 4. 419.6.

o Constitutive promoter+RBS-WbnJ-terminator - 1. 405.4; 2. 76.6; 3. 110.3; 4. 117.7.

o pLacI/Ara-1+RBS-WfcA-terminator - 1. 345.2; 2. 370.7; 3. 392.3; 4. 450.8.

Protocol for adding cultures to liquid medium was followed using chloramphenicol as the selection antibiotic.

Protocol for Mini-Prep was followed exactly as Friday 13th July, except:

o For centrifugation of 5mL culture at 3'900rcf for 10 minutes at 21°C prior to the purification protocol.

o At step 4, 13'100rpm for 5 minutes at 21°C was used.

o At step 10, 40µL of MilliQ H20 was used to elute DNA from the column.

Gel Electrophoresis showed constitutive promoter+RBS-WbnJ-terminator and pLacI/Ara-1+RBS-WfcA-terminator were successfully ligated together. Lane 1 = DNA hyperladder, Lane 2 and lane 5 (1A and 1D) = pLacI/Ara-1+RBS-WfcA-terminator (expected: 2 bands at 1307bp and 2070bp), Lane 6-10 (2A-2D) = TetR+RBS-WbnJ-terminator (expected: 2 bands at 925bp and 2070bp), Lane 12 (9B) = constitutive promoter+RBS-WbnJ-terminator (expected: 2 bands at 906bp and 2070bp).

Wednesday 19th September (9.00)

• Mini-Prepping of OmpA

• Send off OmpA for sequencing

• Transformation of pBAD/AraC promoter weak+RBS-GFP, pBAD/AraC promoter strong+RBS-GFP, pBAD(large)+RBS-GFP, constitutive promoter+RBS-GFP, pLacI/Ara-1+RBS-GFP, WclY-pSB1C3, WbnJ-pSB1C3, WbbC-pSB1C3, WfcA-terminator and RBS-WbiP

• Adding cultures with pBAD(large)+RBS-OmpA-SacB-terminator, TetR+RBS-OmpA-SacB-terminator, TetR+RBS-Has-terminator (x 2), pLacI/Ara-1+RBS-WclY-terminator and TetR+RBS-WbnK-terminator into liquid medium and incubation overnight

The following concentrations (in ng/µL) was obtained:

o OmpA - 1. 45.2.

Protocol for adding cultures to liquid medium was followed using chloramphenicol for pBAD(large)+RBS-OmpA-SacB-terminator and TetR+RBS-OmpA-SacB-terminator, kanamycin for TetR+RBS-Has-terminator (x 2) and TetR+RBS-WbnK-terminator, and ampicillin for pLacI/Ara-1+RBS-WclY-terminator as selection antibiotics.

Thursday 20th September (9.00)

• Making stocks of 2.5µM L-arabinose, 1µM IPTG and 1µg/mL tetracycline

• Inducing of pBAD(large)+RBS-OmpA-SacB-terminator, TetR+RBS-OmpA-SacB-terminator, TetR+RBS-Has-terminator (x 2), pLacI/Ara-1+RBS-WclY-terminator and TetR+RBS-WbnK-terminator to determine protein expression

• Adding cultures with pBAD/AraC promoter weak+RBS-GFP, pBAD/AraC promoter strong+RBS-GFP, pBAD(large)+RBS-GFP, constitutive promoter+RBS-GFP, pLacI/Ara-1+RBS-GFP, WclY-pSB1C3, WbnJ-pSB1C3 and RBS-WbiP into liquid medium and incubation overnight

• Transformation of WbbC and WfcA-terminator

The method for determining protein expression was as follows:

o Two conical flasks were labelled for each full construct being tested for protein expression (hence 12 conical flasks in total). One was labelled uninduced (-) and the other induced (+). The uninduced labelled conical flask was needed to determine basal levels of protein expression.

o 2.5µM L-arabinose was added to + inducer pBAD(large)+RBS-OmpA-SacB-terminator, 1µM IPTG was added to + inducer pLacI/Ara-1+RBS-WclY-terminator and 1µg/mL tetracycline was added to + inducer TetR+RBS-OmpA-SacB-terminator, + inducer TetR+RBS-Has-terminator (x 2) and + inducer TetR+RBS-WbnK-terminator.

o Under sterile conditions, a 1/20 dilution of pBAD(large)+RBS-OmpA-SacB-terminator, TetR+RBS-OmpA-SacB-terminator, TetR+RBS-Has-terminator (x 2), pLacI/Ara-1+RBS-WclY-terminator and TetR+RBS-WbnK-terminator liquid broths were made using LB broth in appropriately labelled conical flasks.

o Each dilution contained a 1000-fold dilution of appropriate antibiotic for each full construct (see selection antibiotics in previous day).

o 750µL was immediately pipetted out of each conical flask (both - inducer and + inducer for every full construct) and transferred to fresh cuvettes and the OD600 was measured for all 12 conical flasks. A LB broth control was used to calibrate the spectrophotometer.

o After all 12 conical flasks had been tested for OD600, they were placed in a shaking incubator set at 37°C at 250rpm.

o After an hour incubating in the shaking incubator, 750µL out of each of the 12 conical flasks was put into fresh cuvettes and the OD600 was measured, again, using an LB broth control.

o Next, 250µL of each of the 12 conical flasks was transferred to appropriately labelled 1.5mL Eppendorf tubes which were labelled 'Pellet'.

o Again, all 12 conical flasks were placed in the shaking incubator at 37°C at 250rpm.

o The 12 1.5mL Eppendorf tubes labelled with 'Pellet' containing 250µL of cultures were centrifuged at 13'100rpm for 5 minutes at 21°C.

o Once centrifuged, the supernatant was transferred to fresh 1.5mL Eppendorf tubes labelled 'Supernatant' for each full construct being tested (both - inducer and + inducer). Dry pellet and supernatant of each - inducer and + inducer of the six full constructs were placed in a -20°C freezer.

o After an hour and a half, this process (from measuring OD600 in 750µL samples to placing dry pellet and supernatant into the -20°C freezer) was repeated and subsequently repeated after another hour and a half, and then at 9:15am the next morning.

The results for each OD600 measured is as follows:

o 1a = 0.357. | o 1a = 0.722. | o 1a = 1.151. | o 1a = 2.451.

o 1b = 0.375. | o 1b = 0.761. | o 1b = 1.156. | o 1b = 2.467.

o 2a = 0.306. | o 2a = 0.752. | o 2a = 1.185. | o 2a = 2.453.

o 2b = 0.178. | o 2b = 0.748. | o 2b = 0.438. | o 2b = 1.287.

o 3a = 0.125. | o 3a = 0.419. | o 3a = 1.020. | o 3a = 1.941.

o 3b = 0.098. | o 3b = 0.172. | o 3b = 0.331. | o 3b = 1.299.

o 4a = 0.304. | o 4a = 0.941. | o 4a = 1.560. | o 4a = 2.239.

o 4b = 0.226. | o 4b = 0.428. | o 4b = 0.732. | o 4b = 2.060.

o 5a = 0.247. | o 5a = 0.877. | o 5a = 2.255. | o 5a = 2.223.

o 5b = 0.240. | o 5b = 0.548. | o 5b = 1.007. | o 5b = 1.684.

o 6a = 0.129. | o 6a = 0.130. | o 6a = 0.259. | o 6a = 2.545.

o 6b = 0.111. | o 6b = 0.101. | o 6b = 0.179. | o 6b = 2.234.

At: 13.30, 15.00, 16.30 and 9.15 the following day from left to right respectively

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1 = pBAD(large)+RBS-OmpA-SacB-terminator

2 = TetR+RBS-OmpA-SacB-terminator

3 = TetR+RBS-Has-terminator 1

4 = TetR+RBS-Has-terminator 2

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

6 = TetR+RBS-WbnK-terminator

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a = uninduced (- inducer)

b = induced (+ inducer)

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No colonies appeared on WbbC-pSB1C3 and WfcA-terminator spreads plates and therefore these were repeated again.

Friday 21st September (9.00)

• Running pellet's and supernatant's of each full construct (both induced and uninduced samples) on SDS-PAGE

• Mini-Prepping of pBAD/AraC promoter weak+RBS-GFP, pLacI/Ara-1+RBS-GFP, WclY-pSB1C3, WbnJ-pSB1C3 and RBS-WbiP.

• Send off WclY-pSB1C3 and WbnJ-pSB1C3 to the Parts Registry.

The following concentrations (in ng/µL) were obtained:

o pBAD/AraC promoter weak+RBS-GFP - 163.1.

o pLacI/Ara-1+RBS-GFP - 129.6.

o WclY-pSB1C3 - 354.8.

o WbnJ-pSB1C3 - 1. 322.2; 2. 298.4.

o RBS-WbiP - 1. 234.4; 2. 143.9; 3. 49.2; 4. 212.1.

The protocol for SDS-PAGE was followed exactly for looking at protein expression in pellets or supernatants of the 6 constructs, using 2µL pellet or supernatant and 3µL MilliQ H20 for loading samples onto the gel. The two gels (one for supernatant, another for pellet) was left to stain for the weekend before visualisation on Monday.

No cultured liquid broths appeared for pBAD/AraC promoter strong+RBS-GFP, constitutive promoter+RBS-GFP and pBAD(large)+RBS-GFP. For RBS-WbiP all the mini-preps were pink due to overcrowding on the Petri dish.