Team:Exeter/lab book/3gip/wk3

From 2012.igem.org

ExiGEM2012 Lab Book 3GP wk3


Protocols \| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase
9th - 13th July - 16th - 20th July - 23rd - 27th July - 30th July - 3rd August - 6th - 10th August - 13th - 17th August - 20th - 24th August - 27th - 31st August - 3rd - 7th September - 10th - 14th September - 17th - 21st September - Results
	The 3-Gene Inducible Plasmid: 23rd - 27th July 2012 Monday 23rd July Morning •Preparation of IDTE buffer Protocol was followed exactly. Tuesday 24th July Morning •3A Assembly BBa_K206001 + BBa_B0034 BBa_K206000 + BBa_B0034 BBa_K094120 + BBa_B0034. All the constructs were ligated into pSB1C3 using the New England Biolab Protocol. The protocol required 500ng of plasmid DNA. Using Linearized Plasmid Backbone this at 25ng/μl this used more plasmid than we hoped. Afternoon • IDT resuspension and transformation of wbnJ and wfcA Alex and Becca finished the transformation of the wbnJ and wfcA genes while Mary and I continued with the BioBrick transformation. 2ul of DNA was used for each transformation into Invitrogen TOP10 competent cells using the protocol provided. Tuesday 25th July Morning The 3A assembly and subsequent transformation performed on Tuesday 24th July showed no colonies in the morning. It was thought the problem may be that the BBa_B0034 sequence was too small (12bp). We decided to run the 3A assembly again but this time alongside the standard assembly allowing us to compare. •3A Assembly The 3A assembly was repeated for: BBa_K206000 + BBa_B0034 into pSB1T3 BBa_K094120 + BBa_B0034 into pSB1T3 • Standard assembly Standard assembly using BBa_B0034 in the destination plasmid. The amount of DNA used was increased to 2000ng and the total H20 was changed from 50μl to 35μl to fit the gel. For the standard assembly the samples were digested for 1 hour at 37°C. A 50μl 8% gel, using low melting agarose, was prepared. While the gel was drying we used a Qiagen Purification Kit to purify the downstream BBa_B0034 digestion. This was performed using the Qiagen Gel Extraction Protocol exactly. Afternoon • Standard assembly Gel Purification The digested upstream DNA, BBa_K206000 and BBa_K094120, were run on the 8% gel. The gel was run for 20 minutes until the dye had run 2 thirds the length of the gel. The gel was taken into the darkroom, visors were worn and lab coats tucked into gloves. The UV tray was pulled out and turned to low. BBa_K206000 and BBa_K094120 showed faint bands but after attempting to cut them out, it was decided that not enough DNA was present for gel purification. •Transformation Products of the 3A assembly: BBa_K206000 + BBa_B0034 BBa_K094120 + BBa_B0034 Using Invitrogen TOP10 competent cells split between 2 eppendorfs so that each contained 25μl Spread on two tetracycline plates for each transformation using 20μl and 100μls respectively. Wednesday 26th July Afternoon •Transformation Pbad promoter BBa_I0500, 2012 Distribution Kit Plate 1, 14N •Transferring colonies to liquid medium Cells containing BBa_K206000 + BBa_B0034 and BBa_K094120 + BBa_B0034 both showed colonies and were added into liquid medium and incubated overnight Protocol was followed using kanamycin for all BioBrick parts as the selection antibiotic. Thursday 27th July Morning Pbad promoter BBa_I0500 showed colonies and was stored in the fridge until Monday. •Miniprepping BBa_K206000_BBa_B0034 BBa_K094120_ BBa_B0034 Both BBa_K206000_BBa_B0034 and BBa_K094120_ BBa_B0034 were nanodropped and recorded good concentrations. •Gel Electrophoresis Run to check fragment sizes of BBa_K206000_BBa_B0034 and BBa_K094120_ BBa_B0034 after digestion using the EcoR1 and Pst1 enzymes Gel Electrophoresis showed no bands but this may have been down to the small size of the inserts. Streak plates were made from the overnight cultures and left on the bench until Monday.

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