Revision as of 02:54, 22 October 2009

Meetings Calendar

Metal Sensing

This sub-project focusses on getting the Bacillus subtilis to sense the cadmium and being able to respond to this detection. Please click on the links below for more information:

Introduction
Modelling
BioBrick Constructs
Lab Work Strategies
Other Presentations and Diagrams
Lab Work done

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. This system of combined metal sensors ensures the Bacillus subtilis cells have a tightly controlled cadmium sensor, as naturally occurring metal sensitive promoters can respond to OTHER metals too

There are two metal sensing repressors, which are known to respond to cadmium: ArsR and CzrA. However, their specificity for cadmium is not unique. ArsR also detects arsenic and CzrA also detects zinc and copper. The table below shows the metals which release both ArsR and CzrA from their DNA binding sites:

Metal Sensor	Metals Sensed
ArsR	As(III)	Ag(I)	Cu	Cd
CzrA	Zn	Co	Ni	Cd

By positioning the operator binding sites for these two metal sensing repressors next to each other in a promoter region, the gene regulated by that promoter will be transcribed only when a metal that binds to both sensors is present; in this case Cadmium.

This is a combinatorial approach for gene expression regulation. To read more information on the cadmium sensor, please visit the Cadmium Sensing section of the Project Overview.

To create this AND gate the team made use of the CadA promoter; this promoter, which usually regulates expression of the CadA cadmium efflux channels in the presence of cadmium ions, bears the CzrA binding site. Therefore if this promoter is coupled to the ArsR binding site, expression of a signal protein will only be made in the presence of cadmium.

Why don't we just use the CadA promoter then? The answer is this - cadA can respond to other metals too so a system totally reliant on the cadA promoter as a sensor would not be efficient in achieving the aims of this project. We need a tightly-controlled cadmium sensor

Modelling

To simulate the effects of cadmium intake and sensing, the Metal Sensing Team constructed models using CellML. Please click on the link here: Metal Intake/Efflux Model

BioBrick constructs

For our cadmium-sensing BioBrick design, we have ligated the ArsR binding site([http://partsregistry.org/wiki/index.php?title=Part:BBa_K174016 BBa_K174016]) next to the CadA promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K174017 BBa_K174017]). The CadA promoter drives expression of the CadA efflux channel and this expression activity increase in the presence of cadmium. Yet more importantly the CadA promoter region contains the CzrA binding site the team needs for the cadmium-sensor AND gate.

By placing the ArsR binding site next to the CadA promoter, expression of a signal protein will only be achieved if there is cadmium in the cell.

Lab Work Strategies

In carrying out the lab work required for the construction of the cadmium-sensor, we adhered to the following lab strategy. Please click on the link here to review our strategy: Metal Sensor: czrA+arsR.

Lab Work done

This is all of the lab work in which the Metal Sensing Team attempted to construct the cadmium sensor - click on the dates to read that day's lab entry:

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
17th September 2009	Subsequent work on cadmium sensor put on hold

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)

Newcastle iGEM Twitter
[http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
[http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]

@@ Line 69: / Line 69: @@
 Please refer the key below to better understand this diagram! In short, cadmium ions make their way through the MntH channel and bind to the ArsR and CzrA repressor proteins. Their removal allows the DNA polymerase to make a signal protein which will feed into the stochastic switch.
 <br>
-[[Image:Team Newcastle 2009 iGEM MetalSensingkey.png|350px|center]]
+[[Image:Team Newcastle MetalSensingkey.png|350px|center]]
 ==Lab Work done==

Team:Newcastle/Metalsensing

From 2009.igem.org