Team:Exeter/lab book/1gp/wk5

Operon Construction  |  Glycobase

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• IDT re-suspension and transformation of WbnK and WclY

Tuesday 7th August (15.00)

• Adding cultures with WbnK and WclY plasmids into liquid medium and incubation overnight

Protocol was followed using ampicillin for WbnK and WclY plasmids as the selection antibiotic.

Wednesday 8th August (9.00)

• Mini-Prepping of WbnK and WclY plasmids

• 3A assembly of WbnK, WclY and terminator

• Transformation of ligated WbnK-terminator and ligated WclY-terminator

The following volumes of 500ng DNA required for 3A assembly were:

o WbnK - 2.42µL (from 206.7ng/µL).

o WclY - 1.61µL (from 309.6ng/µL).

o (Double) terminator - 11.6µL (from 43.1ng/µL)

The first two DNA constructs were cut using EcoRI-HF and SpeI whilst the last DNA construct was cut using XbaI and PstI. The destination plasmid (pSB1C3) was cut using EcoRI-HF and PstI, using the linearized plasmid backbone protocol. Ligation of desired constructs was followed exactly as the protocol stated.

Thursday 9th August (15.00)

• Adding cultures with ligated WbnK-terminator and ligated WclY-terminator plasmids into liquid medium and incubation overnight

Protocol was followed using chloramphenicol for ligated WbnK-terminator and WclY-terminator plasmids as the selection antibiotic.

Friday 10th August (9.00)

• Mini-Prepping of ligated WbnK-terminator and WclY-terminator plasmids

• Gel Electrophoresis to check fragment sizes of WbnJ, WbnK, WclY, WfcA, WbbC(with point mutation), ligated WbnK-terminator, ligated WbbC(with point mutation)-terminator and ligated WclY-terminator

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

o WbnK-terminator - 46.4.

o WclY-terminator - 65.5.

Gel electrophoresis showed that all single gene constructs were correct. However, only WclY-terminator out of the double constructs worked. Lane 1 = DNA hyperladder, Lane 2 = WbnJ (expected: 764bp and 2155bp), Lane 3 = WbnJ-pSB1C3(expected: 2 bands at 764bp and 2070bp), Lane 4 = WfcA (expected: 2 bands at 1097bp and 2155bp), Lane 5 = WbnK (expected: 2 bands at 908bp and 2155bp), Lane 6 = WbnK-terminator (expected: 2 bands at 1003bp and 2070bp), Lane 7 = WclY (expected: 2 bands at 1061bp and 2155bp), Lane 8 = WclY-terminator (expected: 2 bands at 1156bp and 2070bp), Lane 9 = WbbC(with point mutation) (expected: 2 bands at 1113bp and 2155bp), Lane 10 = WbbC(with point mutation)-terminator (expected: 2 bands at 1208bp and 2070bp), Lane 11 = DNA hyperladder.