From 2009.igem.org

(Difference between revisions)

Latest revision as of 02:23, 22 October 2009

Meetings Calendar

Stochastic Switch

Introduction

One of the most exciting aspects of our project is our synthetic stochastic switch. The switch regulates the decision to become a non-germinating metal container spore, or a spore that can go on to germinate as part of the normal life cycle. Whilst stochastic oscillators have been implemented before using transcriptional regulators, our switch makes use of an invertable DNA segment to ensure that the decision is heritable.

By differentially controlling the expression of the Hin invertase, we designed our switch to be tunable to achieve a biased heads or tails response, allowing a range of probabilities of orientation of the invertable segment to be achieved.

Novelty in this sub-project

We designed a synthetic stochastic switch by using an invertible segment of DNA flanked by a pair of promoters. Depending on the orientation of the invertible sequence, coding sequences will be expressed which reflect the decision to be a metal container or not. We also tuned the natural stochasticity of the sporulation system towards greater sporulation rates by altering the rate of Spo0A phosphorylation.

Gfp concentrations. IPTG:[0-9000nM], Xylose=[0-9000nM], Arabinose=1000nM

BioBrick constructs

There are a number of bricks involved within the stochastic switch construct.

The stochastic brick construct uses the Hin invertase system in order to flip a region between Hix sites. The directionality of the promoter determines whether the switch is 'on' or 'off'. When the promoter is facing right it allows transcription of genes that control:

- Prevention of germination
- Upregulation of sporulation rate
- Expression of the metal sponge (SmtA)
- Decreased cadmium efflux
- Upregulation of cadmium import

Importantly, Hin is differentially expressed depending on the levels of the two inducible promoters that flank the invertable segment on which it lies. This means the segment can be biased in a predictable and controllable fashion to favour one orientation or the other.

The following diagram shows our stochastic construct:

Prevention of germination

The prevention of germination is governed by another invertase switch. When the sequence faces right, a FimE protein is expressed which inverts a further promoter region. This promoter controls expression of the cwlD and sleB genes. If their promoter is in the correct orientation then the cell will be able to germinate and continue as a vegetative cell. However if their promoter has been flipped, the cell can not germinate following sporulation, and will be trapped as a metal containing spore.

Upregulation of sporulation rate

The upregulation of sporulation involves increasing KinA expression. kinA codes a kinase protein that phosphorylates the Spo0A protein to its active form. When the promoter region within our stochastic brick faces right, there will be increased KinA expression, and thus a greater sporulation rate.

Metal sponge and cadmium influx/efflux

Our stochastic switch determines whether the spores can germinate, or whether they are commited to be metal containers that cannot germinate again. We need this switch as we cannot totally interrupt the natural life cycle of the bacteria, since a proportion of cells have to go on to seed the next generation. Expression of the metallothionein fusion protein (cotC-gfp-smtA), cadmium import channel (mntH) and the cadmium efflux channel (cadA) is also governed by the direction of the stochastic promoter. When the direction of promoter faces right, the metallothionein fusion protein's expression will be triggered, ant will soak up the cadmium. While the import channel is upregulated, the efflux system's activity will be slowed down to increase the amountof cadmium inside the cell.

Stochastic Brick

We decided to get our stochastic construct synthesised, as trying to build the construct manually would be too time consuming. The following sequencher diagram shows the components of the construct we had synthesised.

Testing construct

In order to test our construct we had to redesign using inducible promoters governing Hin invertase expression. We used the promoters pSpac and pxylA (Induced by IPTG and Xylose) to test our system. We include cut sites around these promoters in order to replace them with SigmaA promoters once the construct has been characterised.(See sequencher diagram above)

Degradation controller

In order to have another level of control over the orientation of the promoter within the flipping region we added a degradation tag to the Hin invertase protein. The following paper describes how proteins including modified ssrA tags can be located to the ClpXP protease by an Sspb protein. This means that inducible Sspb expression can requlate degradation levels of the tagged protein.

[http://www3.interscience.wiley.com/journal/121415079/abstract?CRETRY=1&SRETRY=0 Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP ]

We decided to put the Sspb protein under the control of an arabinose inducible promoter as the following diagram illustrates. We also included a region of the sac gene in our construct, so that the region will integrate into the Bacillus genome at a region other than amyE.

We added a modified version of ssrA degradation tag onto the C-terminus of the Hin protein. Expressed proteins are therefore degraded by ClpXP. However mutations on the ssrA tag weaken the recognition by ClpX, and the modified tags require the SspB adaptor protein to be recognized. When the SspB protein is expressed the proteins tagged with modified version of ssrA tag are targeted for degradation. Otherwise they remain stable.

In B. subtilis there is no sspB orthologue and SspB from E. coli works in B. subtilis. By regulating the levels of SspB by arabinose, we implemented an inducable protein degradation device.

Hin vs SspB according to the speed of degradation by ClpXP

The wild type E. coli ssrA tag is AANDENY-ALAA (SspB recognition site – ClpX recognition site). As suggested in the paper, we took one of the modified ssrA tags to use in our system.

AANDENY-SENY-ALGG (SspB recognition site – SENY +4 Linker - ClpX recognition site)

This tag works well in B. subtilis. However, degradation tags can affect the activity of proteins. Different degradation tags may effect the activity of different proteins. It has been shown that this tag effected the activity of ComA(1).

Griffith, K. L., and A. D. Grossman. 2008. Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol. Microbiol. 70:1012-1025.

Stochastic Modelling Tools

Matlab can be used for stochastic modelling. The Glasgow team used Matlab to implement the Gillespie algorithm for incorporating noise among cells. They also used deterministic modelling using ODEs and compared their results. When the number of cells increase two approaches become similar since the noise is cancelled out.

Stocks 2 is another stochastic simulation tool which also uses Gillespie’s direct method and supports SBML.

We used computational modelling in Matlab to try to determine how to make our system tuneable.

Please see our modelling page for Matlab files on our stochastic switch model.

FimE switch

The FimE switch is a similar switch to the Hix system. However, it acts as a latch, meaning that once flipped the segmant will not flip back.

[http://genomics.lbl.gov/Stuff/TimHam-BandB-online%20version.pdf fimE switch for DNA re-arrangement]

A Tightly Regulated Inducible Expression System Utilising the fim Inversion Recombination Switch.(E. Coli) Timothy S. Ham, Sung Kuk Lee, Jay D. Keasling,Adam P. Arkin,Received 21 December 2005; accepted 2 March 2006 Published online 13 March 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20916

We decided to use FimE to switch off or on the production of a protein of our choice, such as the genes involved in germination.

[http://jb.asm.org/cgi/reprint/183/14/4190?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=subtilis&searchid=1&FIRSTINDEX=880&resourcetype=HWFIG Control of the Arabinose Regulon in Bacillus subtilis by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping]
[http://ukpmc.ac.uk/articlerender.cgi?artid=310841 Binding of the Bacillus subtilis spoIVCA product to the recombination sites of the element interrupting the sigma K-encoding gene] =>...DNA rearrangement that depends on the spoIVCA gene product...

Bistability in Bacillus subtilis

Read this page to find more options for natural stochastic switches in Bacillus subtilis. Natural stochastic switches:Bistability in Bacillus subtilis

And to find out how we are tuning sporulation using our stochastic switch choice see the sporulation tuning page.

Lab strategies

To carry out our labwork we needed cloning strategies for all of our bricks and devices. Please see our cloning strategies page for details on how we cloned our devices.

Summary of lab work success:
Date:	Achievement:
11/09/09	Successfully cloned the sspB degradation controller fragment into pSB1AT3 Lab book
18/09/09	Sucessfully cloned the ara promoter/ operator fragment into pSB1AT3 Lab book
24/09/09	Successfully cloned the sspB fragment into the ara + pSB1AT3 prepared backbone. We now have an arabinose inducible degradation controller! Lab book

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 7: / Line 7: @@
 ==Introduction==
-One of the most unique aspects of our project is our synthetic stochastic switch which regulates the decision to be a metal container spore, or a spore that can go on to germinate as part of the normal life cycle. Whilst stochastic oscillators have been implemented before using transcriptional regulators, our switch makes use of an invertable DNA segment to ensure that the decision is heritable.
+One of the most exciting aspects of our project is our synthetic stochastic switch. The switch regulates the decision to become a non-germinating metal container spore, or a spore that can go on to germinate as part of the normal life cycle. Whilst stochastic oscillators have been implemented before using transcriptional regulators, our switch makes use of an invertable DNA segment to ensure that the decision is heritable.
+By differentially controlling the expression of the Hin invertase, we designed our switch to be tunable to achieve a biased heads or tails response, allowing a range of probabilities of orientation of the invertable segment to be achieved.
+[[Image:Newcastle r Switchv2.gif|center]]
 ==Novelty in this sub-project==
-We intend to design a synthetic stochastic switch by using an invertible segment of our DNA that codes a promoter. Depending on the direction of the promoter, coding sequences will be expressed which reflect the decision to be a metal container or not. We also plan to tune the natural stochasticity of the sporulation system towards greater sporulation rates by altering the rate ''Spo0A'' phosphorylation.
+We designed a synthetic stochastic switch by using an invertible segment of DNA flanked by a pair of promoters. Depending on the orientation of the invertible sequence, coding sequences will be expressed which reflect the decision to be a metal container or not. We also tuned the natural stochasticity of the sporulation system towards greater sporulation rates by altering the rate of ''Spo0A'' phosphorylation.
-[[Image:Team_Newcastle_iGEM_2009_StochasticSwitch_GFP_2.png|350px]]
+[[Image:Team_Newcastle_iGEM_2009_StochasticSwitch_GFP_2.png|thumb|center|350px|Gfp concentrations. IPTG:[0-9000nM], Xylose=[0-9000nM], Arabinose=1000nM]]
 ==BioBrick constructs==
-There will be various bricks involved within the stochastic switch construct, both those involved in the complete system as well as those designed for testing within the lab.
+There are a number of bricks involved within the stochastic switch construct.
-The stochastic brick construct uses the Hin invertase system in order to flip a promoter region between Hix sites. The directionality of the promoter determines whether the switch is 'on' or 'off'. When the promoter is facing right it allows transcription of genes that control:
+The stochastic brick construct uses the Hin invertase system in order to flip a region between Hix sites. The directionality of the promoter determines whether the switch is 'on' or 'off'. When the promoter is facing right it allows transcription of genes that control:
 ** Prevention of germination
 ** Upregulation of sporulation rate
@@ Line 24: / Line 30: @@
 ** Decreased cadmium efflux
 ** Upregulation of cadmium import
+Importantly, Hin is '''differentially expressed''' depending on the levels of the two inducible promoters that flank the invertable segment on which it lies. This means the segment can be '''biased''' in a predictable and controllable fashion to favour one orientation or the other.
 The following diagram shows our stochastic construct:
@@ Line 29: / Line 39: @@
 [[Image:Team NewcastleStochastic switch.png| center|550px]]
-[[Image:Team newc Stoch key.png| center 100px]]
+[[Image:Team newc Stoch key.png| 200px]]
 ===Prevention of germination===
-The prevention of germination is governed by another invertase switch. Once the stochastic promoter faces right, a FimE protein is expressed which inverts a further promoter region. This promoter controls expression of the ''CwlD''
+The prevention of germination is governed by another invertase switch. When the sequence faces right, a FimE protein is expressed which inverts a further promoter region. This promoter controls expression of the ''cwlD''
-and ''SleB'' genes knocked out within our chassis. If their promoter is in the correct orientation then the cell will be able to germinate and continue as a vegetative cell. However if their promoter has been flipped, the cell can not germinate following sporulation, and will be trapped as a metal containing spore.
+and ''sleB'' genes. If their promoter is in the correct orientation then the cell will be able to germinate and continue as a vegetative cell. However if their promoter has been flipped, the cell can not germinate following sporulation, and will be trapped as a metal containing spore.
 ===Upregulation of sporulation rate===
-The upregulation of sporulation involves increasing ''KinA'' expression. ''KinA'' codes a kinase protein that phosphorylates the ''Spo0A'' protein to its active form. When the promoter region within our stochastic brick faces right, there will be increased KinA expression, and thus a greater sporulation rate.
+The upregulation of sporulation involves increasing KinA expression. ''kinA'' codes a kinase protein that phosphorylates the Spo0A protein to its active form. When the promoter region within our stochastic brick faces right, there will be increased KinA expression, and thus a greater sporulation rate.
 ===Metal sponge and cadmium influx/efflux===
-Expression of the metallothionein fusion protein (''CotC-GFP-SmtA''), cadmium import channel (''mntH'') and the canmium efflux channel (CadA) is also governed by the direction of the stochastic promoter.
+Our stochastic switch determines whether the spores can germinate, or whether they are commited to be metal containers that cannot germinate again. We need this switch as we cannot totally interrupt the natural life cycle of the bacteria, since a proportion of cells have to go on to seed the next generation.
+Expression of the metallothionein fusion protein (''cotC-gfp-smtA''), cadmium import channel (''mntH'') and the cadmium efflux channel (''cadA'') is also governed by the direction of the stochastic promoter. When the direction of promoter faces right, the metallothionein fusion protein's expression will be triggered, ant will soak up the cadmium. While the import channel is upregulated, the efflux system's activity will be slowed down to increase the amountof cadmium inside the cell.
 ===Stochastic Brick===
-We have decided to get our stochastic construct synthesised, as trying to build the construct manually would be too time costly. The following sequencher diagram shows the components of the construct we had synthesised.
+We decided to get our stochastic construct synthesised, as trying to build the construct manually would be too time consuming. The following sequencher diagram shows the components of the construct we had synthesised.
-[[Image:Team newc Sequencher synth stoch.png| center|700px]]
+[[Image:Team newc Sequencher synth stoch.png| center|600px]]
 ===Testing construct===
-In order to test our construct we have had to redesign using inducible promoters governing Hin invertase expression. We have used promoters ''pSpac'' and ''pxylA'' (Induced by IPTG and Xylose) to test our system. We include cut sites around these promoters in order to replace them with SigmaA promoters once the construct has been characterised.(See sequencher diagram above)
+In order to test our construct we had to redesign using inducible promoters governing Hin invertase expression. We used the promoters ''pSpac'' and ''pxylA'' (Induced by IPTG and Xylose) to test our system. We include cut sites around these promoters in order to replace them with SigmaA promoters once the construct has been characterised.(See sequencher diagram above)
 ===Degradation controller===
-In order to have another level of control over the orientation of the promoter within the flipping region we have added a degradation tag to the hin invertase protein. The following paper describes how proteins including modified ssrA tags can be located to the ClpXP protease by an sspb protein. This means that inducible sspb expression can requlate degradation levels of the tagged protein.
+In order to have another level of control over the orientation of the promoter within the flipping region we added a degradation tag to the Hin invertase protein. The following paper describes how proteins including modified ''ssrA'' tags can be located to the ClpXP protease by an Sspb protein. This means that inducible Sspb expression can requlate degradation levels of the tagged protein.
-[http://www3.interscience.wiley.com/journal/121415079/abstract?CRETRY=1&SRETRY=0 Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP ]
+[http://www3.interscience.wiley.com/journal/121415079/abstract?CRETRY=1&SRETRY=0 Inducible protein degradation in ''Bacillus subtilis'' using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP ]
-We have decided to put the sspb protein under the control of an arabinose inducible promoter as the following diagram illustrates, also we have included a region of the sac gene in our construct, so that the region will integrate into the ''Bacillus'' genome at a region other than amyE.
+We decided to put the Sspb protein under the control of an arabinose inducible promoter as the following diagram illustrates. We also included a region of the ''sac'' gene in our construct, so that the region will integrate into the ''Bacillus'' genome at a region other than ''amyE''.
 [[Image:Team NewcIntegration Deg control.png |center|500px]]
-We added a modified version of ''ssrA'' degradation tag onto the C-terminus of Hin protein. Hence expressed proteins are degraded by ClpXP. However mutations on the ''ssrA'' tag weakens the recognition by ClpX and the modified tags require sspB adaptor protein to be recognized. Hence when sspB protein is expressed, the proteins tagged with modified version of ''ssrA'' tag are targeted for degradation, otherwise they remain stable.
+We added a modified version of ''ssrA'' degradation tag onto the C-terminus of the Hin protein. Expressed proteins are therefore degraded by ClpXP. However mutations on the ''ssrA'' tag weaken the recognition by ClpX, and the modified tags require the SspB adaptor protein to be recognized. When the SspB protein is expressed the proteins tagged with modified version of ''ssrA'' tag are targeted for degradation. Otherwise they remain stable.
-In ''B. subtilis'' there is no sspB orthologue and SspB from ''E. coli'' works in ''B. subtilis''.  By regulating the levels of sspB by arabinose, we designed an inducable protein degradation device.
+In ''B. subtilis'' there is no ''sspB'' orthologue and SspB from ''E. coli'' works in ''B. subtilis''.  By regulating the levels of SspB by arabinose, we implemented an inducable protein degradation device.
-[[Image:Team_Newcastle_iGEM_2009_Degradation_Model_4.png|400px]]
+[[Image:Team_Newcastle_iGEM_2009_Degradation_Model_4.png|thumb|center|400px|Hin vs SspB according to the speed of degradation by ClpXP]]
-Wild type ''E. coli'' ''ssrA'' tag is '''AANDENY-ALAA''' (SspB recognition site – ClpX recognition site). As suggested in the paper, we took one of the modified ''ssrA'' tags to use in our system.
+The wild type ''E. coli'' ''ssrA'' tag is '''AANDENY-ALAA''' (SspB recognition site – ClpX recognition site). As suggested in the paper, we took one of the modified ''ssrA'' tags to use in our system.
 '''AANDENY-SENY-ALGG''' (SspB recognition site – SENY +4 Linker - ClpX recognition site)
-This tag works well in ''B. subtilis'' however degradation tags can affect activity of proteins. Different degradation tags may have effect on the activity of different proteins. It has been shown that this tag effected the activity of ComA(1).
+This tag works well in ''B. subtilis''. However, degradation tags can affect the activity of proteins. Different degradation tags may effect the activity of different proteins. It has been shown that this tag effected the activity of ComA(1).
-#Griffith, K. L., and A. D. Grossman. 2008. Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol. Microbiol. 70:1012-1025.
+#Griffith, K. L., and A. D. Grossman. 2008. Inducible protein degradation in ''Bacillus subtilis'' using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol. Microbiol. 70:1012-1025.
-We have the following cloning strategies for testing our construct.
-==Lab Work Strategies==
-====Cloning strategies====
-Currently in the lab we are half way to acheiving a complete manual brick which is the degradation controller. In the lab so far, the stochastic switch team have:
-*Rehydrated, transformed, and miniprepped the biiobricks involved in promoter replacement, as well as frozen down these ''E.coli'' strains into the TPA collection.
-*Prepared the pSB1AT3 plasmid backbone (restriction digest, gel extraction) ready for ligation.
-*Have successful PCR products for the sac region (''bacillus'') and sspb (''E.coli''), and a possible success for the ara region.
-* Have done digests for sspb and sac, sspb has been extracted from the gel and is due for cloning.
-==Modelling==
 ===Stochastic Modelling Tools===
-'''Matlab''' can be used for stochastic modelling. Glasgow team used Matlab implementing Gillespie algorithm to incorporate noise among cells. They also used deterministic modelling using ODEs and compared their results. When the number of cells increase two approaches become similar since the noise is cancelled out.
+'''Matlab''' can be used for stochastic modelling. The Glasgow team used Matlab to implement the Gillespie algorithm for incorporating noise among cells. They also used deterministic modelling using ODEs and compared their results. When the number of cells increase two approaches become similar since the noise is cancelled out.
 '''Stocks 2''' is another stochastic simulation tool which also uses Gillespie’s direct method and supports SBML.
-CellML model for the expression of Hin system
-[[Media:flipping.txt]]
+We used computational modelling in Matlab to try to determine how to make our system tuneable.
-We have used computational modelling in Matlab to try to determine how to make our system tuneable.
 Please see our [[Team:Newcastle/Modelling|modelling]] page for Matlab files on our stochastic switch model.
-===Metal Container Decision===
+===FimE switch===
-Our stochastic switch decides whether the spores can germinate, or whether they are commited to be a metal containing spore that cannot germinate again. We need this switch as we cannot interrupt the natural life cycle of the bacteria, as a proportion have to go on to seed the next generation.
+The FimE switch is a similar switch to  the Hix system. However, it acts as a latch, meaning that once flipped the segmant will not flip back.
-We looked at the following possibilities:
-===Hin/Hix system===
-In 2006, Davidson team tried to solve the burnt pancake problem by using DNA rearrangement using Hin/Hix system from ''Salmonella typhimurium''. (http://parts2.mit.edu/wiki/index.php/Davidson_2006.)  Basically they tried to use the bacteria as a biomemory! They also have a paper published which is attached.
-Their animation explains the process quite well.
-(http://www.bio.davidson.edu/people/kahaynes/FAMU_talk/Living_computer.swf)
-The parts they submitted to the parts registry have "W" flag which means they are working.
-http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2006&group=iGEM2006_Davidson
-The Hin system will be the main DNA rearrangement system within our stochastic switch,
-Unlike the ''fim'' system the ''hin'' system allows the DNA segment to be flipped back and forth, therefore a pulse of hin expression is what we would need to ensure we can get the correct proportion to be metal containers.
-The switch as an overall diagram
-[[Media:MetalContainerDecisionSwitch.ppt]]
-Animation of how the switch works
-[[Media:switch animation.ppt]]
-==Other Presentations and Diagrams==
-===fimE switch===
-The FimE switch is a similar switch to  the Hix system, however it acts as a latch, meaning once flipped, the segmant will not flip back.
 # [http://genomics.lbl.gov/Stuff/TimHam-BandB-online%20version.pdf  fimE switch for DNA re-arrangement]
 A Tightly Regulated Inducible Expression System Utilising the fim Inversion Recombination Switch.(''E. Coli'') Timothy S. Ham, Sung Kuk Lee, Jay D. Keasling,Adam P. Arkin,Received 21 December 2005; accepted 2 March 2006 Published online 13 March 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20916
-We could use it switch off or on the production of a protein of our choice, such as the genes involved in germination.
+We decided to use FimE to switch off or on the production of a protein of our choice, such as the genes involved in germination.
 # [http://jb.asm.org/cgi/reprint/183/14/4190?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=subtilis&searchid=1&FIRSTINDEX=880&resourcetype=HWFIG Control of the Arabinose Regulon in Bacillus subtilis by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping]
@@ Line 143: / Line 112: @@
 And to find out how we are tuning sporulation using our stochastic switch choice see the sporulation tuning page.
+===Lab strategies===
+To carry out our labwork we needed cloning strategies for all of our bricks and devices. Please see our [[Team:Newcastle/ Stochastic Switch cloning strategy| cloning strategies]] page for details on how we cloned our devices.
+{|style="color:DarkBlue;background-color:#ffffcc;" cellpadding="20" cellspacing="0" border="1"
+! colspan="2" |<font size=3> <center>'''Summary of lab work success:'''</center></font>
+|-
+|'''Date:'''
+|'''Achievement:'''
+|-
+|11/09/09
+|Successfully cloned the ''sspB'' degradation controller fragment into pSB1AT3
+[[Team:Newcastle/Labwork/11_September_2009 | Lab book]]
+|-
+|18/09/09
+|Sucessfully cloned the ''ara'' promoter/ operator fragment into pSB1AT3
+[[Team:Newcastle/Labwork/18_September_2009 | Lab book]]
+|-
+|24/09/09
+|Successfully cloned the ''sspB'' fragment into the ''ara'' + pSB1AT3 prepared backbone. We now have an arabinose inducible degradation controller!
+[[Team:Newcastle/Labwork/24_September_2009| Lab book]]
+|}
 {{:Team:Newcastle/Footer}}
 {{:Team:Newcastle/Right}}

Team:Newcastle/Stochasticity