Team:Newcastle/Metalsensing

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|'''[https://2009.igem.org/Team:Newcastle/Labwork/7_September_2009#Metal_Sensor_Team 7th September 2009]'''
|Inoculated LB media with colonies of ''BBa_J33206'' (sent from Chris French) ''E. coli'' transformants for mini preps. By afternoon, cultures had grown sufficiently to further inoculate flasks of 50ml LB + amp for midi-preps. Mini-prep attempted but abandoned  
|Inoculated LB media with colonies of ''BBa_J33206'' (sent from Chris French) ''E. coli'' transformants for mini preps. By afternoon, cultures had grown sufficiently to further inoculate flasks of 50ml LB + amp for midi-preps. Mini-prep attempted but abandoned  
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|'''[https://2009.igem.org/Team:Newcastle/Labwork/8_September_2009#Metal_Sensor_Team]'''
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Revision as of 21:51, 21 October 2009



Metal Sensing

Introduction

If our project is to process cadmium and not other metals, we need to genetically engineer Bacillus subtilis to carry out a set of cellular processes based on the action of metal sensors. These metal sensors will detect cadmium through a system known as AND Gating.

There are two metal sensing repressors, which are known to respond to cadmium: arsR and czrA. By placing binding sites to these two metal sensing repressors next to each other in a promoter region, the gene regulated by that promoter will be synthesized only when a combination of metals that bind to both sensors are present; this is a combinatorial approach for gene expression regulation.

Modelling

BioBrick constructs

Lab Work Strategies

Other Presentations and Diagrams

Newcastle Metalsensor2100.gif

Lab Work done

Summary of Lab Sessions for Cadmium Sensing
Date
Description
18th August 2009 Transformed DH5-alpha E. coli cells with BBa_J33206 from the Spring Distribution
19th August 2009 Inoculated 3 tubes of LB with 3 colonies of potential transformant E.coli cells
20th August 2009 Conduct mini-preps on the three overnight-grown cultures of potential BBa_J33206 transformants and digested them with restriction enzymes.
21st August 2009 Analysed digested BBa_J33206 mini-prep DNA by DNA gel electrophoresis and prepared midi-preps also.
25th August 2009 Concentrated (and ethanol precipitated) the BBa_J33206 Midi-prep sample.
26th August 2009 Digested BBa_J33206 midi-prep DNA with EcoRI and PstI and analysed through DNA gel electrophoresis - digest reaction not successful (a second attempt at digests needed)
27th August 2009 Digested pSB1A2 (containing BBa_J33206 BioBrick), ran DNA though gel and excised band. Also analysed digested pSB1A2 (BBa_J33206 BioBrick) in gel - bands erroneous
28th August 2009 Cleaned gel band using Gel extraction kit
1st September 2009 Attempted to PCR amplify the needed czrA gene from the genome of Bacillus subtilis
2nd September 2009 PCR amplification of czrA gene failed - conducted B. subtilis genome prep and carried out PCR reaction on this DNA
3rd September 2009 Second attempt at czrA PCR amplification analysed on gel - also unsuccessful. Reattempted PCR amplification of czrA on B. subtilis genomic DNA at different annealing temperatures.
4th September 2009 In light of 27/08/09 lab session (i.e. anomalous bands with digested BBa_J33206 BioBrick), we have sent away the BioBrick for sequencing and are now using BBa_J33206 sent to us by Chris French. Used Chris's BBa_J33206 to transform E. coli cells. Also carried out PCR reactions
7th September 2009 Inoculated LB media with colonies of BBa_J33206 (sent from Chris French) E. coli transformants for mini preps. By afternoon, cultures had grown sufficiently to further inoculate flasks of 50ml LB + amp for midi-preps. Mini-prep attempted but abandoned
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