Team:Exeter/lab book/1gp/wk3

Operon Construction  |  Glycobase

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• Preparation of IDTE Buffer

Protocol was followed exactly.

Tuesday 24th July (9.00)

• 3A assembly of pBAD/AraC weak-RBS, pBAD/AraC strong-RBS and pLacI/Ara-1

• IDT Re-suspension and transformation of WbnJ and WfcA

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

o pBAD/AraC weak - 2.37µL (from 211.4ng/µL).

o pBAD/AraC strong - 3.83µL (from 130.4ng/µL).

o pLacI/Ara-1 - 1.67µL (from 299.2ng/µL).

o RBS - 6.46µL (from 77.4ng/µL).

The first three 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.

Wednesday 25th July (15.00)

• Adding cultures with WbnJ and WfcA plasmids into liquid medium and incubation overnight

Protocol was followed using ampicillin for WbnJ and WfcA plasmids as the selection antibiotic.

Since 3A assembly had failed in selecting for promoter-RBS recombinant plasmids, 3A assembly was repeated and transformation performed again (See: Week 23rd-27th July 2012, Operon Construction: Mary Beton).

Thursday 26th July (9.00)

• Mini-Prepping of WbnJ and WfcA plasmids

• Gel Electrophoresis to check fragment sizes of WbnJ and WfcA plasmids

• Adding E.coli BL21(DE3) into liquid medium and incubation overnight

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

o WbnJ - 247.2.

o WfcA - 299.8.

WbnJ and WfcA were left to restriction digest with EcoRI-HF and PstI overnight at 37°C.

Because glycosyltransferase WbbC could not be synthesised without a point-mutation present, WbbC will be amplified from the laboratory strain BL21(DE3). The protocol for adding cultures to liquid medium was followed using ampicillin as the selection antibiotic.

Friday 27th July (8.30)

• Genomic extraction from E.coli BL21(DE3)

• PCR amplification of WbbC from E.coli BL21(DE3) using two-step PCR

• Gel Electrophoresis to check fragment size of WbbC PCR product

Protocol for genomic extraction was followed exactly, but at step 9 waiting for 5 minutes to incubate before centrifuging for 1 minute at 6'500 x g and repeating this procedure again.

The following concentration (in ng/µL) obtained for the BL21(DE3) genomic DNA was: 26.9.

The protocol for 2-step PCR using Phusion polymerase was followed exactly.

Gel Electrophoresis did not show a PCR product of the correct size (expected: 1113bp, see below) and it was thought that the first elution of DNA in the Genomic Extraction protocol was discarded. This procedure was repeated again.

Gel Electrophoresis showed that WfcA and WbnJ had been successfully cloned. Lane 1 = DNA hyperladder, Lane 2 = WfcA (expected: 1097bp), Lane 3 = WbnJ (expected: 764bp).