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Team:Newcastle/Labwork/16 September 2009
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====1) Processing the ''cotC-GFP-smtA'' in ''pMUTIN4'' ''E. coli'' transformants==== | ====1) Processing the ''cotC-GFP-smtA'' in ''pMUTIN4'' ''E. coli'' transformants==== | ||
=====a) Mini-preps of ''cotC-GFP-smtA'' ''E. coli'' transformants===== | =====a) Mini-preps of ''cotC-GFP-smtA'' ''E. coli'' transformants===== | ||
| + | The mini-preps of the 12 colonies of ''E. coli'' cells (which are potentially ''cotC-GFP-smtA'' transformants) were carried out using [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=NA0150|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma-Aldrich's GenElute HP Plasmid Mini-prep kit] with it's attached protocol. There were no changes to the protocol | ||
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=====b) Digesting and analysing the mini-preps===== | =====b) Digesting and analysing the mini-preps===== | ||
====2) Second attempt at ligating together cadmium sensor==== | ====2) Second attempt at ligating together cadmium sensor==== | ||
Revision as of 20:49, 12 October 2009
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Formal Lab Session - 16th September 2009
Stochastic Switch Team
Today we ran the ara PCRs from yesterday on a gel and got a really good result, this product was then cleaned, cut with EcoRI and SpeI, cleaned again, then ligated with the pre-cut pSB1AT3 plasmid backbone.
Today we also did minipreps of 12 each of the sac and ara cultures... however unfortunately today the centrifuge we had been relying on broke, and therefore all three teams were forced to use one small centrifuge. This meant that the minipreps could end up being of poor quality due to waiting times for the centrifuge.
Metal Sensing Team
Introduction
In the last lab session, the Metal Sensing picked 12 colonies from a plate containing E. coli + cotC-GFP-smtA transformants and used them to inoculate 12 tubes containing LB + ampicillin - these wil be used for mini-preps today. In yesterday's session we also discovered that the attempts to transform E. coli with the arsR-cadA cadmium sensor failed, so on that same day we carried out a second attempt at transforming E. coli with this BioBrick.
Today's lab session will be focussed on carrying out the mini-preps for the 12 colonies of potential E. coli + cotC-GFP-smtA transformants and based on analysis, will determine whether midi-preps will follow or whether we need to reattempt another transformation (we would, of course, have to carry out another ligation reaction of the cotC BioBrick and the pMUTIN4 plasmid first)
To prepare for the eventuality that the ligations, transformations and midi-preps of the cotC-GFP-smtA in pMUTIN4 worked, the "Evening before transformations day" steps of the Bacillus subtilis transformation protocol will be carried out too.
The plate containing the cadmium-sensor E. coli transformants will also be studied - if there are colonies, 12 of them will be prepared for mini-preps; if there is no success then ligation of the [http://partsregistry.org/Part:BBa_J33206 BBa_J33206 BioBrick] (promoter removed) with the cadA promoter will be carried out - E. coli cells will then be transformed with this.
Practical Outline
- Carry out mini-preps on the 12 colonies of potential E. coli + cotC-GFP-smtA (in pMUTIN4) transformants
- Digest these mini-preps with BamHI and HindIII'
- Analyse the mini-prep digests by DNA gel electrophoresis
- If there are any successes, prepare midi-prep cultures from leftover overnight incubations
- If no colonies present the desired DNA then conduct another ligation of cotC with pMUTIN4 and transform E. coli with this ligated product.
- Carry out the "Evening before transformations" step in the Bacillus subtilis transformation protocol.
- If there are arsR + cadA promoter (cadmium sensor) transformants on LB + ampicillin plate then prepare mini-preps from cultures. If unsuccessful:
- Digest the arsR backbone (BBa_J33206 without promoter) and the cadA promoter with BamHI and NheI
- Carry out overnight ligation reaction
Observations
The only observation that needs to be made now is: there are no colonies on the LB + ampicillin plates where competent E. coli cells, which we had attempted to transform with the BBa_J33206 BioBrick (which contains the arsR binding site and arsR gene) ligated to cadA promoter, were plated. This could be down to three things:
- The ligation between the BBa_J33206 BioBrick (minus promoter) and the cadA promoter didn't work.
- The transformation reaction didn't work
- Both the ligations and the transformations didn't work
Procedure
1) Processing the cotC-GFP-smtA in pMUTIN4 E. coli transformants
a) Mini-preps of cotC-GFP-smtA E. coli transformants
The mini-preps of the 12 colonies of E. coli cells (which are potentially cotC-GFP-smtA transformants) were carried out using [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=NA0150|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sigma-Aldrich's GenElute HP Plasmid Mini-prep kit] with it's attached protocol. There were no changes to the protocol
b) Digesting and analysing the mini-preps
2) Second attempt at ligating together cadmium sensor
a) Digesting the BBa_J33206 backbone and cadA promoter
b) Ligating the BBa_J33206 backbone with cadA promoter
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News
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- 20 – 21 June 2009 - Europe workshop (London)
- 23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
- 23 October Practice Presentation (Newcastle)
- 23 October T-shirts are ready
- 27 October Practice Presentation (Sunderland)
- 27 October Poster is ready
- 30 October – 2 November 2009 - Jamboree (Boston)
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