Team:Exeter/lab book/3gip/wk10

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ExiGEM2012 Lab Book 3GP wk6


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
	The 3-Gene Inducible Plasmid: 10th - 14th September 2012 Monday 10th September Morning •3A assembly BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1) BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2) BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1) BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2) BBa_J23119 + wbnJ BBa_B0014 in pSB1C3 BBa_B0034 + wfcA BBaB0014 in pSB1C3 BBa_B0034 + wfcA BBaB0014 in pSB1K3 BBaK094120_BBa_B0034 + wfcA BBaB0014 in pSB1C3 Performed with the NEB protocol with some adaptions again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a Linearized Plasmid Backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below. Tuesday 11th September Morning •Converting Biobricks into pSB1C3 wbnJ into pSB1C3 wbnK into pSB1C3 wfcA into pSB1C3 wbbC(1) into pSB1C3 wbbC(2) into pSB1C3 wbbC(3) into pSB1C3 wclY into pSB1C3 wbiP into pSB1C3 wbiP into pSB1C3 The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for an hour and a half and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4µl of plasmid DNA was used to 2µl of destination plasmid. Afternoon •Transformation The ligations above and the ligations from the 12/09/2012 were transformed using the Competent Cell protocol and using the competent cells made in the lab. Wednesday 12th September Morning wbbC(1) into pSB1C3, wbbC(2) into pSB1C3, wbbC(3) into pSB1C3 and wbiP into pSB1C3 did not form colonies on the plates. This was repeated. •Converting Biobricks into pSB1C3 ompA into pSB1C3 wbbC(1) into pSB1C3 wbbC(2) into pSB1C3 wbbC(3) into pSB1C3 wbiP into pSB1C3 The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for an hour and a half and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid. •3A assembly ompA + amyA BBa_B0014 Performed with the NEB protocol with some adaptations again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a linearized plasmid backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below. •Two step Phusion PCR using a 6 X 10 serial dilution The reaction was set up using the Biolabs Phusion PCR protocol. Two reactions were set up: Reaction 1: Pbad Large and wbnJ using the primers supplied. See Operon Construction: 9th - 13th July 2012 Reaction 2: Reaction 1: Pbad Large and wbnJ using the primers supplied and varying the magnesium concentration. See Operon Construction: 9th - 13th July 2012 After PCR, PCR mix was dyed, loaded onto the gel and run for 20 minutes. No bands were shown on the gel so the PCR did not work. Afternoon •Adding cultures Cells transformed with below were added into liquid medium and incubated overnight. BBa_E0240 BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1) BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2) BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1) BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2) BBa_J23119 + wbnJ BBa_B0014 in pSB1C3 BBa_B0034 + wfcA BBaB0014 in pSB1C3 BBaK094120_BBa_B0034 + wfcA BBaB0014 in pSB1C3 wbnJ into pSB1C3 wbnK into pSB1C3 wfcA into pSB1C3 wclY into pSB1C3 wbiP into pSB1C3 Protocol was followed using chloramphenicol for the BioBrick part as the selection antibiotic. Thursday 13th. September Morning ompA + amyA BBa_B0014 showed no colonies on the overnight plate. •Mini-Prepping BBa_E0240 BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1) BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2) BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1) BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2) BBa_J23119 + wbnJ BBa_B0014 in pSB1C3 BBa_B0034 + wfcA BBaB0014 in pSB1C3 BBaK094120_BBa_B0034 + WfcA BBaB0014 in pSB1C3 wbnJ into pSB1C3 wbnK into pSB1C3 wfcA into pSB1C3 wclY into pSB1C3 wbiP into pSB1C3 All minipreps were nanodropped showing very good concentrations Afternoon •Gel Digest of DNA from 21/08/2012 A digestion using EcoR1 and PstI was run on all of the miniprepped DNA from today. For the digestion we used Fermentas enzymes instead. The protocol remained the same but we used Fermentas Buffer H instead of the NEB Buffer 2. Friday 14th September Today a digestion ligation was set up to put our remaining Biobricks into pSB1C3. wbnJ BBa_K094120_BBaB0034 in pSB1C3 wclY in pSB1C3 wbbc in pSB1C3 wfcA in pSB1C3 BBaB0034_wbiP in pSB1C3 BBaB0034_wbnK in pSB1C3 BBaB0034_wclY in pSB1C3 BBa_K206000_BBa_B0034 in pSB1C3 BBa_ K206001_BBa_B0034 in pSB1C3 BBa_I0500_BBa_B0034 in pSB1C3 BBa_J23119_ BBa_B0034 in pSB1C3 ompA in pSB1C3 ompA K322921_BBa_B0014 in pSB1C3 BBa_K094120_BBa_B0034_wclY BBa_B0014 in pSB1C3 BBa_J13002 _BBa_B0034_wclY BBa_B0014 in pSB1C3 BBa_J13002 amyA BBa_B0014 in pSB1C3 BBa_J13002 hasA BBa_B0014 in pSB1C3 The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for an hour and a half and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid. For the digestion we used Fermentas enzymes instead. The protocol remained the same but we used Fermentas Buffer H instead of the NEB Buffer 2. There was not enough time to transform any of these this afternoon so after the ligation they were stored at -20°C for the weekend.

@@ Line 149: / Line 149: @@
 <p><i>wbbC</i>(1) into pSB1C3, <i>wbbC</i>(2) into pSB1C3, <i>wbbC</i>(3) into pSB1C3 and <i>wbiP</i> into pSB1C3 did not form colonies on the plates. This was repeated. </p><br>
 <p>•<u>Converting Biobricks into pSB1C3</u></p>
-<p><i>ompA<i> into pSB1C3</p>
+<p><i>ompA</i> into pSB1C3</p>
 <p><i>wbbC</i>(1) into pSB1C3</p>
 <p><i>wbbC</i>(2) into pSB1C3</p>