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• Metal Intake/Efflux
• Metal Sensing
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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

• Metal Intake/Efflux Model

BioBrick constructs

Lab Work Strategies

• Metal Sensor: czrA+arsR - plan A

• Metal Sensor: czrA+arsR - plan B

Other Presentations and Diagrams

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
8th September 2009	Skipped mini-prep re-attempt and immediately carried out midi-prep of BBa_J33206 BioBrick sent by Chris French. Digested sample with EcoRI and PstI and analysed through gel. Successful!
10th September 2009	PCR amplification of the cadA promoter and BBa_J33206 BioBrick (missing promoter)
11th September 2009	Gel analysis shows PCR reactions worked. Tried to proceed with running the cleaned-up PCR products through gel to excise band but ethanol presence stopped us. Will have to reattempt PCR reactions and other subsequent steps.
14th September 2009	Both cadA promoter and BBa_J33206 (with promoter missing) PCR reactions worked! Subsequently excised from gel and cleaned up. Both fragments cut with BamHI and NheI and ligated together. Attempted to transform E. coli with ligated cadmium-sensor.
15th September 2009	No transformant colonies spotted on plates - either ligations or transformations didn't work. Reattempted ligation and transformation in E. coli cells.
16th September 2009	Transformations failed! Reattempted digesting the BBa_J33206 (with no promoter) and cadA promoter with BamHI and NheI and also reattempted ligations