http://2012.igem.org/wiki/index.php?title=Special:Contributions/Aleksandra&feed=atom&limit=50&target=Aleksandra&year=&month=2012.igem.org - User contributions [en]2024-03-29T12:09:19ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Paris_Bettencourt/SuicideTeam:Paris Bettencourt/Suicide2012-10-27T01:33:17Z<p>Aleksandra: /* Results */</p>
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<div id="grouptitle">Suicide System</div><br />
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'''Aims :'''<br />
Implement a kill-switch that features population-level suicide and complete genome degradation. <br />
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'''System :'''<br />
A synthetic toxin-anti-toxin system based on the wild type Colicin E2 operon.<br />
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'''Achievements :'''<br />
We showed that Colicin E2 cells induce cell death in sensitive populations, and that these sensitive populations can be protected by providing them with our engineered immunity protein. <br />
* Construction of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914001 K914001] : pLac-repressilator RBS-Colicin E2 immunity protein<br />
** [http://partsregistry.org/Part:BBa_K914002 K914002] :repressilator RBS-Colicin E2 immunity protein<br />
Part K914001 is well characterized and provides immunity to sensitive cells against the Colicin E2 activity protein. Part K914002 is promoterless and allows users to easily plug in the appropriate promoter for their desired purpose. <br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety XNase]. The aim of this category is to provide users with DNase/RNase parts that can be used for improved kill switches featuring the degradation of genomic material.<br />
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* Partially biobricked sRNA system : <br />
**[http://partsregistry.org/Part:BBa_K914017 K914017] stationary phase promoter Yiagp<br />
**[http://partsregistry.org/Part:BBa_K914016 K914016] coding sequence of Colicin E2<br />
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==Overview==<br />
Our goal is to engineer a synthetic toxin-anti-toxin system from the wild type Colicin E2 (Col E2) operon. This synthetic toxin-anti-toxin system is species specific, allows for population-level suicide, complete genome degradation, and will function on a [https://2012.igem.org/Team:Paris_Bettencourt/Delay tunable delay]. The Col E2 toxin, called the activity protein, is a DNase, meaning that it cleaves DNA, which targets related species of ''E.coli''. The Col E2 anti-toxin, called the immunity protein, binds the Col E2 activity protein with high affinity preventing the activity protein from acting on its own producing bacteria. Our idea is to clone the activity protein and the immunity protein on two different plasmids, called toxin and anti-toxin plasmids, so we can switch on the suicide mechanism by degrading the anti-toxin plasmid with the [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme restriction enzyme system]. To test our system, we cloned the immunity protein under the Lac promoter inside a medium copy plasmid. We tested our system by showing that this inducible system protects the cells from the Col E2 activity protein. The next step will be to place the activity protein under a constitutive promoter inside a low copy plasmid; work which is ongoing.<br />
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<center>[[Image:Toxin3aPB12.gif | 400px]]</center><br />
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===Background===<br />
'''What are Colicins?'''<br />
Bacteria have developed mechanisms to kill other bacteria in order to reduce competition between themselves in the environment [1]. Some strains of ''E. coli'' produce '''lethal proteins''' called colicins which kill sensitive bacteria, including related species of E. coli. Colicinogenic cells contain '''specific immunity''' against their own toxin, and their colicins only effect cells containing specific receptors on their outer membrane surface. Colicins are therefore classified by the receptor to which they bind to, for example, colicins E1-E9 bind to the outer membrane (OM) protein BtuB which mediates the entry of nucleosides, siderphores, and vitamin B12 in the cell. Colicins E1-E9 are also Group A colicins, meaning that they are translocated through the cell envelope by the Tol machinery, whereas Group B colicins are translocated through the cell envelope by the TonB machinery. Colicins have different modes of action, including '''enzymatic activity''' such as DNase (DNA cleaving) activity, and '''pore-forming activity'''. Colicins have three domains, the N-terminal domain functions in translocation through the membrane, the central domain is involved in binding to the OM receptor, and the C-terminal domain contains active (lethal) protein region [1]. We have taken advantage of the modularity of the colicin domains, generating a [https://2012.igem.org/Team:Paris_Bettencourt/SID synthetic import domain] that will allow for new and exciting forms of '''communication''' in ''E.coli''.<br />
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[[File:nrmicro2454-f1.jpeg|frameless|center|500px]]<br />
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<center> Kleanthous C Nature Reviews Microbiology 2010 8, 843-848 </center><br />
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For our project we have selected Colicin E2, which has enzymatic DNase activity. Colicin E2 is ideal because it not only kills sensitive cells on a '''population level''', but also '''destroys genomic material''', preventing the spread of genetically modified parts via horizontal gene transfer. <br />
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In natural systems colicin operons are regulated under the SOS promoter (Stress response). Group A colicins contain type I plasmids. These plasmids are generally 6-10 kb, found in about 20 copies per cell, and can be amplified and mobilized in the presence of a conjugative plasmid. The first gene of the colicin operon is the activity protein, named Colicin ''X'' activity (i.e. cea for Colicin E2) . This is followed by the immunity protein, named Colicin ''X'' immunity (cei), which, like the activity protein, is regulated by the LexA promoter, but also has its own constitutive promoter. This separate promoter is located with in the coding sequence of the cea and allows for constant overproduction of the cei, thereby protecting the producing cell. The last gene codes for the lysis protein, named colicin ''X'' lysis protein (cel), which causes the release of colicin into medium and is responsible for the cell death of the producer. The SOS promoter can be induced by UV light, chemicals, and stress conditions[1]. <br />
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We would like to clone the cea and cei of Colicin E2 onto two separate plasmids, creating toxin and antitoxin plasmids.<br />
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===Bigger Picture===<br />
Our antitoxin plasmid can be combined with the [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme restriction enzyme system] to generate a toxin-antitoxin system that works on a tunable delay depending on the induction of the restriction enzyme. Once the degradation of the antitoxin plasmid containing the immunity protein is initiated, the cell will continue to produce the activity protein, which will digest the cells own genetic material as well as its neighbors and extracellular DNA via its DNase activity. <br />
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<center>Scroll through the thumbnails to see the step by step explanation of our project.</center><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/2/23/ParisB_suicide_1.png" width="150px" /><span><img src="/wiki/images/2/23/ParisB_suicide_1.png" width="500px" /><br /><div id="txtOV">In the initial phase, the colicin activity protein and immunity protein are produced and form a heterodimer, protecting the cell from the activity proteins' DNase domain.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/9/93/ParisB_suicide_2.png" width="150px" /><span><img src="/wiki/images/9/93/ParisB_suicide_2.png" width="500px" /><br /><div id="txtOV">In the second phase, the restriction enzyme is produced.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/8/80/ParisB_suicide_3.png" width="150px"/><span><img src="/wiki/images/8/80/ParisB_suicide_3.png" width="500px"/><br /><div id="txtOV">In the third phase, the restriction enzyme recognizes and cleaves the restriction sites present in the antitoxin plasmid.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/4/4f/ParisB_suicide_4.png" width="150px" /><span><img src="/wiki/images/4/4f/ParisB_suicide_4.png" width="500px" /><br /><div id="txtOV">In the fourth phase, the antitoxin plasmid is completely degraded rendering the cell vulnerable to the lethal activity protein.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/f/f5/ParisB_suicide_5.png" width="150px" /><span><img src="/wiki/images/f/f5/ParisB_suicide_5.png" width="500px" /><br /><div id="txtOV">In the fifth phase, the activity protein kills the cell and digests its genetic contents.</div></span></a><br />
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==Objectives==<br />
Bacteria have developed mechanisms to kill other bacteria in order to reduce competition between themselves in their environment [1]. Some strains of ''E. coli'' produce '''lethal proteins''' called colicins which kill sensitive bacteria, including related species of E. coli. Colicinogenic cells contain '''specific immunity''' against their own toxin, and their colicins only effect cells containing specific receptors on their outer membrane surface.<br />
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We have exploited this naturally occurring system to construct a well characterized toxin-antitoxin system ([https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Design design]):<br />
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#Design an antitoxin part from the colicin E2 immunity gene that confers immunity against the Col E2 activity protein<br />
#Measure viability of immune and vulnerable cells in the presence of WT Col E2 cells<br />
#Design a Col E2 activity protein part under the control of a constitutive promoter and establish in immune strains to generate synthetic Col E2 cells<br />
#Measure viability of immune and vulnerable cells in the presence of synthetic Col E2 cells<br />
#Combine our system with the Restriction Enzyme System, measure viability<br />
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==Design==<br />
For our system we will generate two plasmids, one carrying the Col E2 activity protein and one carrying the Col E2 immunity protein. As the immunity protein is normally present in excess in natural colicinogenic cells, we chose a 10-12 copy vector pSB3C5 to carry the immunity protein, and a lower ~5 copy vector pSB4K5 to carry the toxin protein. We used pLac to drive expression of the immunity protein, however, different inducible promoters should be used depending on the overall design and application of the safety circuit. We chose the constitutive promoter BBa_J23108 from the Anderson Promoter Collection, which has a relatively moderate expression level, to drive the expression of the activity protein so that it would not overwhelm the cells until after the desired delay. We ultimately plan to integrate the activity protein cassette into the genome of our bacteria.<br />
[[Image:pSG008.jpg | 500px | center]]<br />
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We do not require the colicin lysis protein for our system, and while the activity protein may degrade extracellular DNA released from our genetically modified bacteria, it is preferable that the cells do not lyse and the genomic material is digested within the cells. A small percentage of cells will die naturally and release the activity protein, which will have a lethal action on any cells that escape our system via mutation of the activity protein gene.<br />
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==Experiments and results==<br />
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===Colicin E2 Expression Kills Sensitive Cells===<br />
We performed a basic assay to characterize the toxicity of the Col E2 cells. The Col E2 cells secrete the lethal activity protein, which diffuses out from the Col E2 colonies spotted on the LB plates. This generates a region where vulnerable cells can not grow, visible as an empty ring (ZOI) around the Col E2 colonies. Here we expect ZOI when Col E2 is spotted on vulnerable cells. <br />
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====Experimental setup====<br />
'''Cell Types'''<br />
*MG1655: These cells approximate wild type E.coli cells as they have undergone minimal genetic manipulation as compared to other laboratory strains. Genotype: F- λ- ilvG- rfb-50 rph-1<br />
*MG1655.RFP: MG1655 cells transformed with constitutive RFP (from Anderson promoter library, [http://partsregistry.org/wiki/index.php/Part:BBa_J23102 BBa_J23102])<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2]<br />
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'''Protocol'''<br />
#Plate 10 uL of 0.1 OD lawn cells from liquid culture on plain LB plates<br />
#Spot 5 uL of saturated Colicin E2 cells from liquid culture on plates<br />
#Incubate plates overnight<br />
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====Results====<br />
Col E2 cells produce ZOI in sensitive cells, as indicated bellow by red arrows. MG1655 and MG1655.RFP, cells which are not colicinogenic, do not produce ZOI when spotted on a lawn of sensitive cells. <br />
[[Image:ParisB_SG_assay.jpg |center]]<br />
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'''Interpretation'''<br />
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The Col E2 cells secrete the lethal activity protein, which diffuses out from the Col E2 colonies spotted on the LB plates. This generates a region where vulnerable cells can not grow, visible as an empty ring (ZOI) around the Col E2 colonies. It is important to note that Col E2 cells do not need to be induced to produce sufficient activity protein to generate the ZOI. These results confirm the toxicity of Col E2 cells against MG1655 and MG1655.RFP, related species of ''E.coli''. We also assayed Top10 cells, NEB turbo cells, MAGE cells, and MG1655 ZI.RFP cells, for more details look<br />
[http://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Notebooks/suicide_group/day_by_day//2012/08/07 here]<br />
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===Expression of Immunity Protects Sensitive Cells===<br />
To test the function of our antitoxin plasmid, we performed Colicin E2 toxicity assays on cells transformed with our plasmid. Here we expect zones of inhibition (ZOI) when Col E2 is spotted on vulnerable cells, and the absence of ZOI in the transformed immune cells.<br />
====Experimental setup====<br />
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'''Cell Types'''<br />
*MG1655: These cells approximate wild type E.coli cells as they have undergone minimal genetic manipulation as compared to other laboratory strains. Genotype: F- λ- ilvG- rfb-50 rph-1<br />
*MG1655.RFP: MG1655 cells transformed with constitutive RFP (from Anderson promoter library, BBa_J61002)<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2] <br />
*NEB.Imm: NEB turbo cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]). This strain is commonly used in the lab as it grows rapidly (visible colonies on agar, ~6.5 hours; shaking liquid culture OD 600 = 2.0, ~4 hours). This strain is also T1 phage resistant and importantly it expresses the Lac repressor (lacR), which allows us to induce the production of the immunity protein the pLac promoter with IPTG. Genotype: F´ proA+B+ lacIq ∆ lacZ M15/ fhuA2 ∆(lac-proAB) glnV gal R(zgb-210::Tn10)TetS endA1 thi-1 ∆(hsdS-mcrB)5<br />
References:New England Biolabs, product catalogue number C2984H<br />
*Z1.Imm: MG1655 Z1 cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]). MG1655 Z1 contain the Z1 cassette (lacR tetR SpR), which allows us to induce the production of the immunity protein under control of the pLac promoter with IPTG.<br />
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'''Protocol'''<br />
#Grow cells in liquid culture containing the appropriate antibiotic<br />
#Add IPTG (0.1mM) to appropriate liquid cultures after 2 hours (OD ~0.2) <br />
#Wash cells containing antibiotics/IPTG and re-suspend in plain LB<br />
#Plate 10 uL of 0.1 OD lawn cells from liquid culture on plain LB plates or IPTG plates<br />
#Spot 5 uL of saturated Colicin E2 cells from liquid culture on plates<br />
#Incubate plates overnight<br />
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====Results====<br />
Col E2 cells do not generate a ZOI in cells containing the antitoxin plasmid, in both induced and un-induced cells. However, Col E2 cells do produce a ZOI in vulnerable MG1655 cells as indicated by red arrows below. <br />
[[Image:SGassayIMM2012.jpg |800px | center]]<br />
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'''Interpretation'''<br />
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These results indicate that our antitoxin plasmid indeed confers immunity to sensitive cells, however, immunity is not dependent on IPTG induction. This is not completely surprising given that the immunity protein has an incredibly high affinity for the activity protein and that pLac is known to be leaky. Even a small concentration of immunity protein could protect un-induced cells against the DNase activity of the colicin activity protein.<br />
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===Quantitative Characterization of the Antitoxin Plasmid===<br />
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We characterized the plasmid pLac + ColE2 immunity gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]) by testing the response of the cells when we mix them with colicin producing cells in various concentration with also various concentration of IPTG as the inducer of pLac. We counted the number of surviving colonies to see the effectiveness of the immunity protein in protecting the cells from the colicin activity protein. <br />
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====Experimental Setup====<br />
'''Cell types'''<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2] <br />
*NEB.CmR: NEB turbo cells transformed with [http://partsregistry.org/Part:pSB3C5 pSB3C5]).<br />
*NEB.Imm: NEB turbo cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]).<br />
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'''Protocol'''<br />
#Grow overnight cultures of NEB.CmR, NEB.Imm each with antibiotic (Cm) and Col E2.<br />
#Centrifuge and resuspend in LB.<br />
#Prepare 5 tubes of 10ml LB and put 40uL of NEB.Imm cells into 4 tubes and NEB.CmR cells in 1 tube.<br />
#Incubate 37C until the OD reaches 0.2.<br />
#Put IPTG with different concentration in 3 NEB.Imm tubes (0.1mM, 0.05mM, 0.025mM).<br />
#Incubate again until the OD reaches 1.0.<br />
#Take each 0.5ml and mix with 0.5ml Colicin cells (saturated, saturated diluted 1000x, plain LB with corresponding amount of IPTG) in small 2ml eppendorf tubes.<br />
#Incubate for 30 minutes.<br />
#Dilute 10000x (100x then 100x) in MgSO4 e-2M.<br />
#Plate 20uL in Cm to kill the Colicin cells and leave the immune cells.<br />
Note: 1ml of cells of OD 1.0 has 1e9 cells, so we expect if all cells survive we will get 1000 colonies at the end.<br />
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====Results====<br />
<!--<br />
The table shows the number of colonies survive in each plate IPTG x Colicin. <br />
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| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| <br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 100uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 10uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 0 IPTG<br />
| style="border:0.035cm solid #000000;padding:0.097cm;"| pSB3C5 (-) control<br />
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|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1x Colicin<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 94<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 79<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 73<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 42<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 0<br />
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|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1000x Colicin<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 9<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 24<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 26<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 74<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 0<br />
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|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| No Colicin (+) control<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 36<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 28<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 30<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 18<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 27<br />
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[[Image:Colicin-assay.png |600px]] </center><br />
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<br />
In this experiment we expected to see more survival if we induce the cells with more IPTG and/or mix them with less Colicin cells. Given that from the previous experiment the immunity protein turned out to be independent to IPTG induction, we treated all the NEB.Imm data as the same. The results showed that the immunity protein can indeed protect the cells from saturated colicin. If we do not put the Colicin cells in the mixture, the numbers of survival cells of NEB.Imm (BBa_K914001) and NEB.CmR (pSB3C5) are almost the same. But if we put the Colicin cells, even very diluted, the NEB.CmR cells could not survive.<br />
<br />
We noticed that the survival on the positive controls (mixture without Colicin) was somehow less than the one with Colicin cells. We guessed that the problems are<br />
*The plates have more cells when they have more Colicin: the Colicin cells maybe took the Immunity/Cm-resistance plasmid<br />
*Less cells on the plate without Colicin (and very few cells overall compared to the theoretical calculation): the cells may have died because they loose the Immunity/Cm-resistance plasmid during the incubation without antibiotic (which we did to avoid killing the Colicin cells).<br />
Therefore we still need to improve protocol to provide a more reliable quantitative data.<br />
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<div id="boston"><br />
===Testing the effects of colicin-containing protein extract on E.coli===<br />
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We mix Z1 cells with the supernatant of centrifuged Colicin cells (exctract with the ColE2 protein). Therefore we can put the antibiotic during the whole experiment to avoid plasmid loss.<br />
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====Experimental Setup====<br />
'''Cell types'''<br />
*colicin-producing cells<br />
*Z1 cells with immunity<br />
*Z1 cells without immunity as a control<br />
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'''Protocol'''<br />
#Grow overnight cultures of colicin-producing cells, Z1 cells with immunity and Z1 cells without immunity.<br />
#For the colicin-producing cells (protocol adapted from http://www.piercenet.com/browse.cfm?fldID=06010401 ):<br />
## Centrifuge at 5000g for 10 mins<br />
## Weigh the falcons with the cell pellet and add 4mL of B-PER Reagent per gram of cell pellet. Pipette the suspension up and down until it is homogeneous<br />
##Incubate 10-15 minutes at room temperature<br />
## Pour the lysate into eppendorff tubes and centrifuge it at 15,000 × g for 5 minutes to separate soluble proteins from the insoluble proteins. Keep the soluble fraction: this is the protein extract containing colicins.<br />
<br />
#For the immune and non-immune cells:<br />
## Dilute the overnight cultures 1/100 into 10-ml cultures (with antibiotics):<br />
### 3 x Z1 cells without immunity<br />
### 3 x Z1 cells with immunity, induced with IPTG<br />
### 3 x Z1 cells with immunity, non-induced<br />
## Grow the cultures for at least 1h, make sure that the OD is more or less the same for all cultures (tip: you can keep a culture with a higher OD at 4°C to wait until the other cultures reach the same OD)<br />
## Mix the immune/non-immune cells with the colicin extract, in different proportions (cells/colicin extract 9:1 and 5:5). For controls, mix immune/non-immune cells with the B-PER buffer, in same proportions<br />
# Put the cells into a 96 well plate for Tecan, 2 wells per each of the cultures<br />
# Put the plate into Tecan, and make OD measurements each 10mins<br />
<br />
====Results====<br />
[[Image:PB2012_Immunity_exp.jpeg|700px|center]]<br />
Growth curves of cells with or without protein extract of colicin producing cells.<br />
We compare the growth of cells that have immunity protein or not. Colicin-containing protein extract significantly impedes the growth of cells that do not have immunity (green curve), whereas it does not affect cells expressing immunity (blue curve), and they grow as well as positive controls (red and purple lines, respectively).<br />
<br />
Remarks: Immune cells grow even better in the presence of the colicin extract than in the presence of the buffer. This can be explained by the fact that the buffer used for protein extraction leads to cell lysis. In future, we will use the extract of cells that do not produce colicin, as a positive control. Also, in this setup, the step-like trend of the growth curve for the sensitive cells in the presence of colicin extract (blue) can be explained by the degradation of the colicin, and/or by the fact that it all colicin present could be used up at some point, and not affect further growth of cells. <br />
<br />
In conclusion, these results show that colicin produced by cells prevents the growth or even kills sensitive cells, whereas the cells expressing the immunity protein survive.<br />
<br />
</div><br />
<br />
====Persperctives====<br />
We suggest in the future we could give stronger stress to the Colicin to induce more the SOS promoter; such as UV light.<br />
<br />
==Discussion==<br />
Cloning the colicin E2 activity protein, given its lethal nature, is a great challenge for us. We took several approaches. We established immune strains expressing excess immunity protein in which we could transform the toxin plasmid. We tried cloning the toxin without a promoter and finally tried various vectors with and with out promoters. Although the immune strains are viable, transformable, and prove immune to wild type colicin E2 cells, we have yet to successfully transform a single toxin plasmid. <br />
<br />
However, developing and characterizing the immune strains was not as straight forward as we imagined. Whether in NEB turbo or MG1655 Z1 cells, the immune strains grew incredibly slowly. Despite being under the control of pLac, their immunity proved somewhat independent of IPTG, as both un-induced and induced cells prove immune. In addition, we had some strange toxicity assay results, partially resolved by the explanation of colicin E2 contamination. <br />
<br />
To see more details on the toxicity assays, click [http://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Notebooks/suicide_group/day_by_day//2012/09/08 here]<br />
<br />
'''UPDATE:'''<br />
We are still struggling with cloning the colicin E2 activity protein, however we have managed to clone the coding sequence and have some more promising results using the sRNA approach described [https://2012.igem.org/Team:Paris_Bettencourt/Delay here]<br />
<br />
==Related Parts and Links==<br />
*[http://partsregistry.org/Part:BBa_K131000 BBa_K131000] Colicin E2 operon, designed by Kevin McLeod, group: iGEM08_Calgary_Wetware (2008-07-22)<br />
*[http://partsregistry.org/Part:BBa_R0011 BBa_R0011] Promoter (lacI regulated, lambda pL hybrid), designed by Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton, group: Registry (2003-01-31)<br />
*[http://partsregistry.org/Part:BBa_R0011:Experience/iGEM10_Kyoto BBa_R0011:Experience/iGEM10 Kyoto] pLac model<br />
*[http://partsregistry.org/pSB4K5 pSB4K5 ] Low copy plasmid containing kanamycin resistance<br />
*[http://partsregistry.org/pSB3C5 pSB3C5 ] Low-medium copy plasmid containing chloramphenicol resistance<br />
*[http://partsregistry.org/wiki/index.php/Part:BBa_J23108 BBa_J23108] Anderson Constitutive Promoter with relative strength 0.51 (RFP reporter)<br />
*[http://partsregistry.org/wiki/index.php/Part:BBa_J23102 BBa_J23102] Anderson Constitutive Promoter with relative strength 0.86 (RFP reporter)<br />
*[http://partsregistry.org/Part:BBa_K914016 K914016] coding sequence of Colicin E2<br />
<br />
==References==<br />
#Cascales E, et al. (2007) Colicin biology. Microbiol Mol Biol Rev 71:158–229. <br />
#Majeed G, Gillor O, Kerr B, Riley MA. (2011) Competitive interactions in Escherichia coli populations: the role of bacteriocins. The ISME Journal 5, 71-81.<br />
#Pugsley AP. (1985) Escherichia coli K12 strains for use in the identification and characterication of colicins. J Gen Microbiol 131: 369-376.<br />
#Kleanthous C. (2010) Swimming against the tide: progress and challenges in our understanding of colicin translocation. Nat Microbiol Rev 8:843-848.<br />
#Torres B, et al. (2003) A dual lethal system to enhance containment of recombinant micr-organisms. Microbiol 149: 3595-3601.<br />
<br />
==Appendix==<br />
===ColicinE2 Plasmid Map===<br />
[[File:ColE2P9.jpg|frameless|center|500px]]<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/SuicideTeam:Paris Bettencourt/Suicide2012-10-27T01:32:59Z<p>Aleksandra: /* Results */</p>
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<br />
<div id="grouptitle">Suicide System</div><br />
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'''Aims :'''<br />
Implement a kill-switch that features population-level suicide and complete genome degradation. <br />
<br />
'''System :'''<br />
A synthetic toxin-anti-toxin system based on the wild type Colicin E2 operon.<br />
<br />
'''Achievements :'''<br />
We showed that Colicin E2 cells induce cell death in sensitive populations, and that these sensitive populations can be protected by providing them with our engineered immunity protein. <br />
* Construction of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914001 K914001] : pLac-repressilator RBS-Colicin E2 immunity protein<br />
** [http://partsregistry.org/Part:BBa_K914002 K914002] :repressilator RBS-Colicin E2 immunity protein<br />
Part K914001 is well characterized and provides immunity to sensitive cells against the Colicin E2 activity protein. Part K914002 is promoterless and allows users to easily plug in the appropriate promoter for their desired purpose. <br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety XNase]. The aim of this category is to provide users with DNase/RNase parts that can be used for improved kill switches featuring the degradation of genomic material.<br />
<div id="boston"><br />
* Partially biobricked sRNA system : <br />
**[http://partsregistry.org/Part:BBa_K914017 K914017] stationary phase promoter Yiagp<br />
**[http://partsregistry.org/Part:BBa_K914016 K914016] coding sequence of Colicin E2<br />
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==Overview==<br />
Our goal is to engineer a synthetic toxin-anti-toxin system from the wild type Colicin E2 (Col E2) operon. This synthetic toxin-anti-toxin system is species specific, allows for population-level suicide, complete genome degradation, and will function on a [https://2012.igem.org/Team:Paris_Bettencourt/Delay tunable delay]. The Col E2 toxin, called the activity protein, is a DNase, meaning that it cleaves DNA, which targets related species of ''E.coli''. The Col E2 anti-toxin, called the immunity protein, binds the Col E2 activity protein with high affinity preventing the activity protein from acting on its own producing bacteria. Our idea is to clone the activity protein and the immunity protein on two different plasmids, called toxin and anti-toxin plasmids, so we can switch on the suicide mechanism by degrading the anti-toxin plasmid with the [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme restriction enzyme system]. To test our system, we cloned the immunity protein under the Lac promoter inside a medium copy plasmid. We tested our system by showing that this inducible system protects the cells from the Col E2 activity protein. The next step will be to place the activity protein under a constitutive promoter inside a low copy plasmid; work which is ongoing.<br />
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<center>[[Image:Toxin3aPB12.gif | 400px]]</center><br />
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===Background===<br />
'''What are Colicins?'''<br />
Bacteria have developed mechanisms to kill other bacteria in order to reduce competition between themselves in the environment [1]. Some strains of ''E. coli'' produce '''lethal proteins''' called colicins which kill sensitive bacteria, including related species of E. coli. Colicinogenic cells contain '''specific immunity''' against their own toxin, and their colicins only effect cells containing specific receptors on their outer membrane surface. Colicins are therefore classified by the receptor to which they bind to, for example, colicins E1-E9 bind to the outer membrane (OM) protein BtuB which mediates the entry of nucleosides, siderphores, and vitamin B12 in the cell. Colicins E1-E9 are also Group A colicins, meaning that they are translocated through the cell envelope by the Tol machinery, whereas Group B colicins are translocated through the cell envelope by the TonB machinery. Colicins have different modes of action, including '''enzymatic activity''' such as DNase (DNA cleaving) activity, and '''pore-forming activity'''. Colicins have three domains, the N-terminal domain functions in translocation through the membrane, the central domain is involved in binding to the OM receptor, and the C-terminal domain contains active (lethal) protein region [1]. We have taken advantage of the modularity of the colicin domains, generating a [https://2012.igem.org/Team:Paris_Bettencourt/SID synthetic import domain] that will allow for new and exciting forms of '''communication''' in ''E.coli''.<br />
<br />
[[File:nrmicro2454-f1.jpeg|frameless|center|500px]]<br />
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<center> Kleanthous C Nature Reviews Microbiology 2010 8, 843-848 </center><br />
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For our project we have selected Colicin E2, which has enzymatic DNase activity. Colicin E2 is ideal because it not only kills sensitive cells on a '''population level''', but also '''destroys genomic material''', preventing the spread of genetically modified parts via horizontal gene transfer. <br />
<br />
In natural systems colicin operons are regulated under the SOS promoter (Stress response). Group A colicins contain type I plasmids. These plasmids are generally 6-10 kb, found in about 20 copies per cell, and can be amplified and mobilized in the presence of a conjugative plasmid. The first gene of the colicin operon is the activity protein, named Colicin ''X'' activity (i.e. cea for Colicin E2) . This is followed by the immunity protein, named Colicin ''X'' immunity (cei), which, like the activity protein, is regulated by the LexA promoter, but also has its own constitutive promoter. This separate promoter is located with in the coding sequence of the cea and allows for constant overproduction of the cei, thereby protecting the producing cell. The last gene codes for the lysis protein, named colicin ''X'' lysis protein (cel), which causes the release of colicin into medium and is responsible for the cell death of the producer. The SOS promoter can be induced by UV light, chemicals, and stress conditions[1]. <br />
<br />
We would like to clone the cea and cei of Colicin E2 onto two separate plasmids, creating toxin and antitoxin plasmids.<br />
<br />
===Bigger Picture===<br />
Our antitoxin plasmid can be combined with the [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme restriction enzyme system] to generate a toxin-antitoxin system that works on a tunable delay depending on the induction of the restriction enzyme. Once the degradation of the antitoxin plasmid containing the immunity protein is initiated, the cell will continue to produce the activity protein, which will digest the cells own genetic material as well as its neighbors and extracellular DNA via its DNase activity. <br />
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<center>Scroll through the thumbnails to see the step by step explanation of our project.</center><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/2/23/ParisB_suicide_1.png" width="150px" /><span><img src="/wiki/images/2/23/ParisB_suicide_1.png" width="500px" /><br /><div id="txtOV">In the initial phase, the colicin activity protein and immunity protein are produced and form a heterodimer, protecting the cell from the activity proteins' DNase domain.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/9/93/ParisB_suicide_2.png" width="150px" /><span><img src="/wiki/images/9/93/ParisB_suicide_2.png" width="500px" /><br /><div id="txtOV">In the second phase, the restriction enzyme is produced.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/8/80/ParisB_suicide_3.png" width="150px"/><span><img src="/wiki/images/8/80/ParisB_suicide_3.png" width="500px"/><br /><div id="txtOV">In the third phase, the restriction enzyme recognizes and cleaves the restriction sites present in the antitoxin plasmid.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/4/4f/ParisB_suicide_4.png" width="150px" /><span><img src="/wiki/images/4/4f/ParisB_suicide_4.png" width="500px" /><br /><div id="txtOV">In the fourth phase, the antitoxin plasmid is completely degraded rendering the cell vulnerable to the lethal activity protein.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/f/f5/ParisB_suicide_5.png" width="150px" /><span><img src="/wiki/images/f/f5/ParisB_suicide_5.png" width="500px" /><br /><div id="txtOV">In the fifth phase, the activity protein kills the cell and digests its genetic contents.</div></span></a><br />
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==Objectives==<br />
Bacteria have developed mechanisms to kill other bacteria in order to reduce competition between themselves in their environment [1]. Some strains of ''E. coli'' produce '''lethal proteins''' called colicins which kill sensitive bacteria, including related species of E. coli. Colicinogenic cells contain '''specific immunity''' against their own toxin, and their colicins only effect cells containing specific receptors on their outer membrane surface.<br />
<br />
We have exploited this naturally occurring system to construct a well characterized toxin-antitoxin system ([https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Design design]):<br />
<br />
#Design an antitoxin part from the colicin E2 immunity gene that confers immunity against the Col E2 activity protein<br />
#Measure viability of immune and vulnerable cells in the presence of WT Col E2 cells<br />
#Design a Col E2 activity protein part under the control of a constitutive promoter and establish in immune strains to generate synthetic Col E2 cells<br />
#Measure viability of immune and vulnerable cells in the presence of synthetic Col E2 cells<br />
#Combine our system with the Restriction Enzyme System, measure viability<br />
<br />
==Design==<br />
For our system we will generate two plasmids, one carrying the Col E2 activity protein and one carrying the Col E2 immunity protein. As the immunity protein is normally present in excess in natural colicinogenic cells, we chose a 10-12 copy vector pSB3C5 to carry the immunity protein, and a lower ~5 copy vector pSB4K5 to carry the toxin protein. We used pLac to drive expression of the immunity protein, however, different inducible promoters should be used depending on the overall design and application of the safety circuit. We chose the constitutive promoter BBa_J23108 from the Anderson Promoter Collection, which has a relatively moderate expression level, to drive the expression of the activity protein so that it would not overwhelm the cells until after the desired delay. We ultimately plan to integrate the activity protein cassette into the genome of our bacteria.<br />
[[Image:pSG008.jpg | 500px | center]]<br />
<br />
We do not require the colicin lysis protein for our system, and while the activity protein may degrade extracellular DNA released from our genetically modified bacteria, it is preferable that the cells do not lyse and the genomic material is digested within the cells. A small percentage of cells will die naturally and release the activity protein, which will have a lethal action on any cells that escape our system via mutation of the activity protein gene.<br />
<br />
==Experiments and results==<br />
<br />
===Colicin E2 Expression Kills Sensitive Cells===<br />
We performed a basic assay to characterize the toxicity of the Col E2 cells. The Col E2 cells secrete the lethal activity protein, which diffuses out from the Col E2 colonies spotted on the LB plates. This generates a region where vulnerable cells can not grow, visible as an empty ring (ZOI) around the Col E2 colonies. Here we expect ZOI when Col E2 is spotted on vulnerable cells. <br />
<br />
====Experimental setup====<br />
'''Cell Types'''<br />
*MG1655: These cells approximate wild type E.coli cells as they have undergone minimal genetic manipulation as compared to other laboratory strains. Genotype: F- λ- ilvG- rfb-50 rph-1<br />
*MG1655.RFP: MG1655 cells transformed with constitutive RFP (from Anderson promoter library, [http://partsregistry.org/wiki/index.php/Part:BBa_J23102 BBa_J23102])<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2]<br />
<br />
'''Protocol'''<br />
#Plate 10 uL of 0.1 OD lawn cells from liquid culture on plain LB plates<br />
#Spot 5 uL of saturated Colicin E2 cells from liquid culture on plates<br />
#Incubate plates overnight<br />
<br />
====Results====<br />
Col E2 cells produce ZOI in sensitive cells, as indicated bellow by red arrows. MG1655 and MG1655.RFP, cells which are not colicinogenic, do not produce ZOI when spotted on a lawn of sensitive cells. <br />
[[Image:ParisB_SG_assay.jpg |center]]<br />
<br />
'''Interpretation'''<br />
<br />
The Col E2 cells secrete the lethal activity protein, which diffuses out from the Col E2 colonies spotted on the LB plates. This generates a region where vulnerable cells can not grow, visible as an empty ring (ZOI) around the Col E2 colonies. It is important to note that Col E2 cells do not need to be induced to produce sufficient activity protein to generate the ZOI. These results confirm the toxicity of Col E2 cells against MG1655 and MG1655.RFP, related species of ''E.coli''. We also assayed Top10 cells, NEB turbo cells, MAGE cells, and MG1655 ZI.RFP cells, for more details look<br />
[http://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Notebooks/suicide_group/day_by_day//2012/08/07 here]<br />
<br />
===Expression of Immunity Protects Sensitive Cells===<br />
To test the function of our antitoxin plasmid, we performed Colicin E2 toxicity assays on cells transformed with our plasmid. Here we expect zones of inhibition (ZOI) when Col E2 is spotted on vulnerable cells, and the absence of ZOI in the transformed immune cells.<br />
====Experimental setup====<br />
<br />
'''Cell Types'''<br />
*MG1655: These cells approximate wild type E.coli cells as they have undergone minimal genetic manipulation as compared to other laboratory strains. Genotype: F- λ- ilvG- rfb-50 rph-1<br />
*MG1655.RFP: MG1655 cells transformed with constitutive RFP (from Anderson promoter library, BBa_J61002)<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2] <br />
*NEB.Imm: NEB turbo cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]). This strain is commonly used in the lab as it grows rapidly (visible colonies on agar, ~6.5 hours; shaking liquid culture OD 600 = 2.0, ~4 hours). This strain is also T1 phage resistant and importantly it expresses the Lac repressor (lacR), which allows us to induce the production of the immunity protein the pLac promoter with IPTG. Genotype: F´ proA+B+ lacIq ∆ lacZ M15/ fhuA2 ∆(lac-proAB) glnV gal R(zgb-210::Tn10)TetS endA1 thi-1 ∆(hsdS-mcrB)5<br />
References:New England Biolabs, product catalogue number C2984H<br />
*Z1.Imm: MG1655 Z1 cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]). MG1655 Z1 contain the Z1 cassette (lacR tetR SpR), which allows us to induce the production of the immunity protein under control of the pLac promoter with IPTG.<br />
<br />
'''Protocol'''<br />
#Grow cells in liquid culture containing the appropriate antibiotic<br />
#Add IPTG (0.1mM) to appropriate liquid cultures after 2 hours (OD ~0.2) <br />
#Wash cells containing antibiotics/IPTG and re-suspend in plain LB<br />
#Plate 10 uL of 0.1 OD lawn cells from liquid culture on plain LB plates or IPTG plates<br />
#Spot 5 uL of saturated Colicin E2 cells from liquid culture on plates<br />
#Incubate plates overnight<br />
<br />
====Results====<br />
Col E2 cells do not generate a ZOI in cells containing the antitoxin plasmid, in both induced and un-induced cells. However, Col E2 cells do produce a ZOI in vulnerable MG1655 cells as indicated by red arrows below. <br />
[[Image:SGassayIMM2012.jpg |800px | center]]<br />
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<br />
'''Interpretation'''<br />
<br />
These results indicate that our antitoxin plasmid indeed confers immunity to sensitive cells, however, immunity is not dependent on IPTG induction. This is not completely surprising given that the immunity protein has an incredibly high affinity for the activity protein and that pLac is known to be leaky. Even a small concentration of immunity protein could protect un-induced cells against the DNase activity of the colicin activity protein.<br />
<br />
===Quantitative Characterization of the Antitoxin Plasmid===<br />
<br />
We characterized the plasmid pLac + ColE2 immunity gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]) by testing the response of the cells when we mix them with colicin producing cells in various concentration with also various concentration of IPTG as the inducer of pLac. We counted the number of surviving colonies to see the effectiveness of the immunity protein in protecting the cells from the colicin activity protein. <br />
<br />
====Experimental Setup====<br />
'''Cell types'''<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2] <br />
*NEB.CmR: NEB turbo cells transformed with [http://partsregistry.org/Part:pSB3C5 pSB3C5]).<br />
*NEB.Imm: NEB turbo cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]).<br />
<br />
'''Protocol'''<br />
#Grow overnight cultures of NEB.CmR, NEB.Imm each with antibiotic (Cm) and Col E2.<br />
#Centrifuge and resuspend in LB.<br />
#Prepare 5 tubes of 10ml LB and put 40uL of NEB.Imm cells into 4 tubes and NEB.CmR cells in 1 tube.<br />
#Incubate 37C until the OD reaches 0.2.<br />
#Put IPTG with different concentration in 3 NEB.Imm tubes (0.1mM, 0.05mM, 0.025mM).<br />
#Incubate again until the OD reaches 1.0.<br />
#Take each 0.5ml and mix with 0.5ml Colicin cells (saturated, saturated diluted 1000x, plain LB with corresponding amount of IPTG) in small 2ml eppendorf tubes.<br />
#Incubate for 30 minutes.<br />
#Dilute 10000x (100x then 100x) in MgSO4 e-2M.<br />
#Plate 20uL in Cm to kill the Colicin cells and leave the immune cells.<br />
Note: 1ml of cells of OD 1.0 has 1e9 cells, so we expect if all cells survive we will get 1000 colonies at the end.<br />
<br />
====Results====<br />
<!--<br />
The table shows the number of colonies survive in each plate IPTG x Colicin. <br />
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| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 100uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 10uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 0 IPTG<br />
| style="border:0.035cm solid #000000;padding:0.097cm;"| pSB3C5 (-) control<br />
<br />
|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1x Colicin<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 94<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 79<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 73<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 42<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 0<br />
<br />
|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1000x Colicin<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 9<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 24<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 26<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 74<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 0<br />
<br />
|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| No Colicin (+) control<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 36<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 28<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 30<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 18<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 27<br />
<br />
|}<br />
<br />
--><br />
<center><br />
[[Image:Colicin-assay.png |600px]] </center><br />
<br />
<br />
In this experiment we expected to see more survival if we induce the cells with more IPTG and/or mix them with less Colicin cells. Given that from the previous experiment the immunity protein turned out to be independent to IPTG induction, we treated all the NEB.Imm data as the same. The results showed that the immunity protein can indeed protect the cells from saturated colicin. If we do not put the Colicin cells in the mixture, the numbers of survival cells of NEB.Imm (BBa_K914001) and NEB.CmR (pSB3C5) are almost the same. But if we put the Colicin cells, even very diluted, the NEB.CmR cells could not survive.<br />
<br />
We noticed that the survival on the positive controls (mixture without Colicin) was somehow less than the one with Colicin cells. We guessed that the problems are<br />
*The plates have more cells when they have more Colicin: the Colicin cells maybe took the Immunity/Cm-resistance plasmid<br />
*Less cells on the plate without Colicin (and very few cells overall compared to the theoretical calculation): the cells may have died because they loose the Immunity/Cm-resistance plasmid during the incubation without antibiotic (which we did to avoid killing the Colicin cells).<br />
Therefore we still need to improve protocol to provide a more reliable quantitative data.<br />
<br />
<div id="boston"><br />
===Testing the effects of colicin-containing protein extract on E.coli===<br />
<br />
We mix Z1 cells with the supernatant of centrifuged Colicin cells (exctract with the ColE2 protein). Therefore we can put the antibiotic during the whole experiment to avoid plasmid loss.<br />
<br />
====Experimental Setup====<br />
'''Cell types'''<br />
*colicin-producing cells<br />
*Z1 cells with immunity<br />
*Z1 cells without immunity as a control<br />
<br />
'''Protocol'''<br />
#Grow overnight cultures of colicin-producing cells, Z1 cells with immunity and Z1 cells without immunity.<br />
#For the colicin-producing cells (protocol adapted from http://www.piercenet.com/browse.cfm?fldID=06010401 ):<br />
## Centrifuge at 5000g for 10 mins<br />
## Weigh the falcons with the cell pellet and add 4mL of B-PER Reagent per gram of cell pellet. Pipette the suspension up and down until it is homogeneous<br />
##Incubate 10-15 minutes at room temperature<br />
## Pour the lysate into eppendorff tubes and centrifuge it at 15,000 × g for 5 minutes to separate soluble proteins from the insoluble proteins. Keep the soluble fraction: this is the protein extract containing colicins.<br />
<br />
#For the immune and non-immune cells:<br />
## Dilute the overnight cultures 1/100 into 10-ml cultures (with antibiotics):<br />
### 3 x Z1 cells without immunity<br />
### 3 x Z1 cells with immunity, induced with IPTG<br />
### 3 x Z1 cells with immunity, non-induced<br />
## Grow the cultures for at least 1h, make sure that the OD is more or less the same for all cultures (tip: you can keep a culture with a higher OD at 4°C to wait until the other cultures reach the same OD)<br />
## Mix the immune/non-immune cells with the colicin extract, in different proportions (cells/colicin extract 9:1 and 5:5). For controls, mix immune/non-immune cells with the B-PER buffer, in same proportions<br />
# Put the cells into a 96 well plate for Tecan, 2 wells per each of the cultures<br />
# Put the plate into Tecan, and make OD measurements each 10mins<br />
<br />
====Results====<br />
[[Image:PB2012_Immunity_exp.jpeg|700px|center]]<br />
Growth curves of cells with or without protein extract of colicin producing cells.<br />
We compare the growth of cells that have immunity protein or not. Colicin-containing protein extract significantly impedes the growth of cells that do not have immunity (green curve), whereas it does not affect cells expressing immunity (blue curve), and they grow as well as positive controls (red and purple lines, respectively).<br />
<br />
Remarks: Immune cells grow even better in the presence of the colicin extract than in the presence of the buffer. This can be explained by the fact that the buffer used for protein extraction leads to cell lysis. In future, we will use the extract of cells that do not produce colicin, as a positive control. Also, in this setup, the step-like trend of the growth curve for the sensitive cells in the presence of colicin extract (blue) can be explained by the degradation of the colicin, and/or by the fact that it all colicin present could be used up at some point, and not affect further growth of cells. <br />
<br />
In conclusion, these results show that colicin produced by cells prevents the growth or even kills sensitive cells, whereas the cells expressing the immunity protein survive.<br />
<br />
====Persperctives====<br />
We suggest in the future we could give stronger stress to the Colicin to induce more the SOS promoter; such as UV light.<br />
<br />
==Discussion==<br />
Cloning the colicin E2 activity protein, given its lethal nature, is a great challenge for us. We took several approaches. We established immune strains expressing excess immunity protein in which we could transform the toxin plasmid. We tried cloning the toxin without a promoter and finally tried various vectors with and with out promoters. Although the immune strains are viable, transformable, and prove immune to wild type colicin E2 cells, we have yet to successfully transform a single toxin plasmid. <br />
<br />
However, developing and characterizing the immune strains was not as straight forward as we imagined. Whether in NEB turbo or MG1655 Z1 cells, the immune strains grew incredibly slowly. Despite being under the control of pLac, their immunity proved somewhat independent of IPTG, as both un-induced and induced cells prove immune. In addition, we had some strange toxicity assay results, partially resolved by the explanation of colicin E2 contamination. <br />
<br />
To see more details on the toxicity assays, click [http://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Notebooks/suicide_group/day_by_day//2012/09/08 here]<br />
<br />
'''UPDATE:'''<br />
We are still struggling with cloning the colicin E2 activity protein, however we have managed to clone the coding sequence and have some more promising results using the sRNA approach described [https://2012.igem.org/Team:Paris_Bettencourt/Delay here]<br />
<br />
==Related Parts and Links==<br />
*[http://partsregistry.org/Part:BBa_K131000 BBa_K131000] Colicin E2 operon, designed by Kevin McLeod, group: iGEM08_Calgary_Wetware (2008-07-22)<br />
*[http://partsregistry.org/Part:BBa_R0011 BBa_R0011] Promoter (lacI regulated, lambda pL hybrid), designed by Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton, group: Registry (2003-01-31)<br />
*[http://partsregistry.org/Part:BBa_R0011:Experience/iGEM10_Kyoto BBa_R0011:Experience/iGEM10 Kyoto] pLac model<br />
*[http://partsregistry.org/pSB4K5 pSB4K5 ] Low copy plasmid containing kanamycin resistance<br />
*[http://partsregistry.org/pSB3C5 pSB3C5 ] Low-medium copy plasmid containing chloramphenicol resistance<br />
*[http://partsregistry.org/wiki/index.php/Part:BBa_J23108 BBa_J23108] Anderson Constitutive Promoter with relative strength 0.51 (RFP reporter)<br />
*[http://partsregistry.org/wiki/index.php/Part:BBa_J23102 BBa_J23102] Anderson Constitutive Promoter with relative strength 0.86 (RFP reporter)<br />
*[http://partsregistry.org/Part:BBa_K914016 K914016] coding sequence of Colicin E2<br />
<br />
==References==<br />
#Cascales E, et al. (2007) Colicin biology. Microbiol Mol Biol Rev 71:158–229. <br />
#Majeed G, Gillor O, Kerr B, Riley MA. (2011) Competitive interactions in Escherichia coli populations: the role of bacteriocins. The ISME Journal 5, 71-81.<br />
#Pugsley AP. (1985) Escherichia coli K12 strains for use in the identification and characterication of colicins. J Gen Microbiol 131: 369-376.<br />
#Kleanthous C. (2010) Swimming against the tide: progress and challenges in our understanding of colicin translocation. Nat Microbiol Rev 8:843-848.<br />
#Torres B, et al. (2003) A dual lethal system to enhance containment of recombinant micr-organisms. Microbiol 149: 3595-3601.<br />
<br />
==Appendix==<br />
===ColicinE2 Plasmid Map===<br />
[[File:ColE2P9.jpg|frameless|center|500px]]<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/SuicideTeam:Paris Bettencourt/Suicide2012-10-27T01:32:14Z<p>Aleksandra: /* Results */</p>
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<br />
<div id="grouptitle">Suicide System</div><br />
<br />
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<td><br />
'''Aims :'''<br />
Implement a kill-switch that features population-level suicide and complete genome degradation. <br />
<br />
'''System :'''<br />
A synthetic toxin-anti-toxin system based on the wild type Colicin E2 operon.<br />
<br />
'''Achievements :'''<br />
We showed that Colicin E2 cells induce cell death in sensitive populations, and that these sensitive populations can be protected by providing them with our engineered immunity protein. <br />
* Construction of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914001 K914001] : pLac-repressilator RBS-Colicin E2 immunity protein<br />
** [http://partsregistry.org/Part:BBa_K914002 K914002] :repressilator RBS-Colicin E2 immunity protein<br />
Part K914001 is well characterized and provides immunity to sensitive cells against the Colicin E2 activity protein. Part K914002 is promoterless and allows users to easily plug in the appropriate promoter for their desired purpose. <br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety XNase]. The aim of this category is to provide users with DNase/RNase parts that can be used for improved kill switches featuring the degradation of genomic material.<br />
<div id="boston"><br />
* Partially biobricked sRNA system : <br />
**[http://partsregistry.org/Part:BBa_K914017 K914017] stationary phase promoter Yiagp<br />
**[http://partsregistry.org/Part:BBa_K914016 K914016] coding sequence of Colicin E2<br />
</div><br />
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<br />
<br />
<br />
<br />
<br />
==Overview==<br />
Our goal is to engineer a synthetic toxin-anti-toxin system from the wild type Colicin E2 (Col E2) operon. This synthetic toxin-anti-toxin system is species specific, allows for population-level suicide, complete genome degradation, and will function on a [https://2012.igem.org/Team:Paris_Bettencourt/Delay tunable delay]. The Col E2 toxin, called the activity protein, is a DNase, meaning that it cleaves DNA, which targets related species of ''E.coli''. The Col E2 anti-toxin, called the immunity protein, binds the Col E2 activity protein with high affinity preventing the activity protein from acting on its own producing bacteria. Our idea is to clone the activity protein and the immunity protein on two different plasmids, called toxin and anti-toxin plasmids, so we can switch on the suicide mechanism by degrading the anti-toxin plasmid with the [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme restriction enzyme system]. To test our system, we cloned the immunity protein under the Lac promoter inside a medium copy plasmid. We tested our system by showing that this inducible system protects the cells from the Col E2 activity protein. The next step will be to place the activity protein under a constitutive promoter inside a low copy plasmid; work which is ongoing.<br />
<br />
<center>[[Image:Toxin3aPB12.gif | 400px]]</center><br />
<br />
===Background===<br />
'''What are Colicins?'''<br />
Bacteria have developed mechanisms to kill other bacteria in order to reduce competition between themselves in the environment [1]. Some strains of ''E. coli'' produce '''lethal proteins''' called colicins which kill sensitive bacteria, including related species of E. coli. Colicinogenic cells contain '''specific immunity''' against their own toxin, and their colicins only effect cells containing specific receptors on their outer membrane surface. Colicins are therefore classified by the receptor to which they bind to, for example, colicins E1-E9 bind to the outer membrane (OM) protein BtuB which mediates the entry of nucleosides, siderphores, and vitamin B12 in the cell. Colicins E1-E9 are also Group A colicins, meaning that they are translocated through the cell envelope by the Tol machinery, whereas Group B colicins are translocated through the cell envelope by the TonB machinery. Colicins have different modes of action, including '''enzymatic activity''' such as DNase (DNA cleaving) activity, and '''pore-forming activity'''. Colicins have three domains, the N-terminal domain functions in translocation through the membrane, the central domain is involved in binding to the OM receptor, and the C-terminal domain contains active (lethal) protein region [1]. We have taken advantage of the modularity of the colicin domains, generating a [https://2012.igem.org/Team:Paris_Bettencourt/SID synthetic import domain] that will allow for new and exciting forms of '''communication''' in ''E.coli''.<br />
<br />
[[File:nrmicro2454-f1.jpeg|frameless|center|500px]]<br />
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<center> Kleanthous C Nature Reviews Microbiology 2010 8, 843-848 </center><br />
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<br />
For our project we have selected Colicin E2, which has enzymatic DNase activity. Colicin E2 is ideal because it not only kills sensitive cells on a '''population level''', but also '''destroys genomic material''', preventing the spread of genetically modified parts via horizontal gene transfer. <br />
<br />
In natural systems colicin operons are regulated under the SOS promoter (Stress response). Group A colicins contain type I plasmids. These plasmids are generally 6-10 kb, found in about 20 copies per cell, and can be amplified and mobilized in the presence of a conjugative plasmid. The first gene of the colicin operon is the activity protein, named Colicin ''X'' activity (i.e. cea for Colicin E2) . This is followed by the immunity protein, named Colicin ''X'' immunity (cei), which, like the activity protein, is regulated by the LexA promoter, but also has its own constitutive promoter. This separate promoter is located with in the coding sequence of the cea and allows for constant overproduction of the cei, thereby protecting the producing cell. The last gene codes for the lysis protein, named colicin ''X'' lysis protein (cel), which causes the release of colicin into medium and is responsible for the cell death of the producer. The SOS promoter can be induced by UV light, chemicals, and stress conditions[1]. <br />
<br />
We would like to clone the cea and cei of Colicin E2 onto two separate plasmids, creating toxin and antitoxin plasmids.<br />
<br />
===Bigger Picture===<br />
Our antitoxin plasmid can be combined with the [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme restriction enzyme system] to generate a toxin-antitoxin system that works on a tunable delay depending on the induction of the restriction enzyme. Once the degradation of the antitoxin plasmid containing the immunity protein is initiated, the cell will continue to produce the activity protein, which will digest the cells own genetic material as well as its neighbors and extracellular DNA via its DNase activity. <br />
<br />
<center>Scroll through the thumbnails to see the step by step explanation of our project.</center><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/2/23/ParisB_suicide_1.png" width="150px" /><span><img src="/wiki/images/2/23/ParisB_suicide_1.png" width="500px" /><br /><div id="txtOV">In the initial phase, the colicin activity protein and immunity protein are produced and form a heterodimer, protecting the cell from the activity proteins' DNase domain.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/9/93/ParisB_suicide_2.png" width="150px" /><span><img src="/wiki/images/9/93/ParisB_suicide_2.png" width="500px" /><br /><div id="txtOV">In the second phase, the restriction enzyme is produced.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/8/80/ParisB_suicide_3.png" width="150px"/><span><img src="/wiki/images/8/80/ParisB_suicide_3.png" width="500px"/><br /><div id="txtOV">In the third phase, the restriction enzyme recognizes and cleaves the restriction sites present in the antitoxin plasmid.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/4/4f/ParisB_suicide_4.png" width="150px" /><span><img src="/wiki/images/4/4f/ParisB_suicide_4.png" width="500px" /><br /><div id="txtOV">In the fourth phase, the antitoxin plasmid is completely degraded rendering the cell vulnerable to the lethal activity protein.</div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/f/f5/ParisB_suicide_5.png" width="150px" /><span><img src="/wiki/images/f/f5/ParisB_suicide_5.png" width="500px" /><br /><div id="txtOV">In the fifth phase, the activity protein kills the cell and digests its genetic contents.</div></span></a><br />
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==Objectives==<br />
Bacteria have developed mechanisms to kill other bacteria in order to reduce competition between themselves in their environment [1]. Some strains of ''E. coli'' produce '''lethal proteins''' called colicins which kill sensitive bacteria, including related species of E. coli. Colicinogenic cells contain '''specific immunity''' against their own toxin, and their colicins only effect cells containing specific receptors on their outer membrane surface.<br />
<br />
We have exploited this naturally occurring system to construct a well characterized toxin-antitoxin system ([https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Design design]):<br />
<br />
#Design an antitoxin part from the colicin E2 immunity gene that confers immunity against the Col E2 activity protein<br />
#Measure viability of immune and vulnerable cells in the presence of WT Col E2 cells<br />
#Design a Col E2 activity protein part under the control of a constitutive promoter and establish in immune strains to generate synthetic Col E2 cells<br />
#Measure viability of immune and vulnerable cells in the presence of synthetic Col E2 cells<br />
#Combine our system with the Restriction Enzyme System, measure viability<br />
<br />
==Design==<br />
For our system we will generate two plasmids, one carrying the Col E2 activity protein and one carrying the Col E2 immunity protein. As the immunity protein is normally present in excess in natural colicinogenic cells, we chose a 10-12 copy vector pSB3C5 to carry the immunity protein, and a lower ~5 copy vector pSB4K5 to carry the toxin protein. We used pLac to drive expression of the immunity protein, however, different inducible promoters should be used depending on the overall design and application of the safety circuit. We chose the constitutive promoter BBa_J23108 from the Anderson Promoter Collection, which has a relatively moderate expression level, to drive the expression of the activity protein so that it would not overwhelm the cells until after the desired delay. We ultimately plan to integrate the activity protein cassette into the genome of our bacteria.<br />
[[Image:pSG008.jpg | 500px | center]]<br />
<br />
We do not require the colicin lysis protein for our system, and while the activity protein may degrade extracellular DNA released from our genetically modified bacteria, it is preferable that the cells do not lyse and the genomic material is digested within the cells. A small percentage of cells will die naturally and release the activity protein, which will have a lethal action on any cells that escape our system via mutation of the activity protein gene.<br />
<br />
==Experiments and results==<br />
<br />
===Colicin E2 Expression Kills Sensitive Cells===<br />
We performed a basic assay to characterize the toxicity of the Col E2 cells. The Col E2 cells secrete the lethal activity protein, which diffuses out from the Col E2 colonies spotted on the LB plates. This generates a region where vulnerable cells can not grow, visible as an empty ring (ZOI) around the Col E2 colonies. Here we expect ZOI when Col E2 is spotted on vulnerable cells. <br />
<br />
====Experimental setup====<br />
'''Cell Types'''<br />
*MG1655: These cells approximate wild type E.coli cells as they have undergone minimal genetic manipulation as compared to other laboratory strains. Genotype: F- λ- ilvG- rfb-50 rph-1<br />
*MG1655.RFP: MG1655 cells transformed with constitutive RFP (from Anderson promoter library, [http://partsregistry.org/wiki/index.php/Part:BBa_J23102 BBa_J23102])<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2]<br />
<br />
'''Protocol'''<br />
#Plate 10 uL of 0.1 OD lawn cells from liquid culture on plain LB plates<br />
#Spot 5 uL of saturated Colicin E2 cells from liquid culture on plates<br />
#Incubate plates overnight<br />
<br />
====Results====<br />
Col E2 cells produce ZOI in sensitive cells, as indicated bellow by red arrows. MG1655 and MG1655.RFP, cells which are not colicinogenic, do not produce ZOI when spotted on a lawn of sensitive cells. <br />
[[Image:ParisB_SG_assay.jpg |center]]<br />
<br />
'''Interpretation'''<br />
<br />
The Col E2 cells secrete the lethal activity protein, which diffuses out from the Col E2 colonies spotted on the LB plates. This generates a region where vulnerable cells can not grow, visible as an empty ring (ZOI) around the Col E2 colonies. It is important to note that Col E2 cells do not need to be induced to produce sufficient activity protein to generate the ZOI. These results confirm the toxicity of Col E2 cells against MG1655 and MG1655.RFP, related species of ''E.coli''. We also assayed Top10 cells, NEB turbo cells, MAGE cells, and MG1655 ZI.RFP cells, for more details look<br />
[http://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Notebooks/suicide_group/day_by_day//2012/08/07 here]<br />
<br />
===Expression of Immunity Protects Sensitive Cells===<br />
To test the function of our antitoxin plasmid, we performed Colicin E2 toxicity assays on cells transformed with our plasmid. Here we expect zones of inhibition (ZOI) when Col E2 is spotted on vulnerable cells, and the absence of ZOI in the transformed immune cells.<br />
====Experimental setup====<br />
<br />
'''Cell Types'''<br />
*MG1655: These cells approximate wild type E.coli cells as they have undergone minimal genetic manipulation as compared to other laboratory strains. Genotype: F- λ- ilvG- rfb-50 rph-1<br />
*MG1655.RFP: MG1655 cells transformed with constitutive RFP (from Anderson promoter library, BBa_J61002)<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2] <br />
*NEB.Imm: NEB turbo cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]). This strain is commonly used in the lab as it grows rapidly (visible colonies on agar, ~6.5 hours; shaking liquid culture OD 600 = 2.0, ~4 hours). This strain is also T1 phage resistant and importantly it expresses the Lac repressor (lacR), which allows us to induce the production of the immunity protein the pLac promoter with IPTG. Genotype: F´ proA+B+ lacIq ∆ lacZ M15/ fhuA2 ∆(lac-proAB) glnV gal R(zgb-210::Tn10)TetS endA1 thi-1 ∆(hsdS-mcrB)5<br />
References:New England Biolabs, product catalogue number C2984H<br />
*Z1.Imm: MG1655 Z1 cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]). MG1655 Z1 contain the Z1 cassette (lacR tetR SpR), which allows us to induce the production of the immunity protein under control of the pLac promoter with IPTG.<br />
<br />
'''Protocol'''<br />
#Grow cells in liquid culture containing the appropriate antibiotic<br />
#Add IPTG (0.1mM) to appropriate liquid cultures after 2 hours (OD ~0.2) <br />
#Wash cells containing antibiotics/IPTG and re-suspend in plain LB<br />
#Plate 10 uL of 0.1 OD lawn cells from liquid culture on plain LB plates or IPTG plates<br />
#Spot 5 uL of saturated Colicin E2 cells from liquid culture on plates<br />
#Incubate plates overnight<br />
<br />
====Results====<br />
Col E2 cells do not generate a ZOI in cells containing the antitoxin plasmid, in both induced and un-induced cells. However, Col E2 cells do produce a ZOI in vulnerable MG1655 cells as indicated by red arrows below. <br />
[[Image:SGassayIMM2012.jpg |800px | center]]<br />
<br />
<br />
'''Interpretation'''<br />
<br />
These results indicate that our antitoxin plasmid indeed confers immunity to sensitive cells, however, immunity is not dependent on IPTG induction. This is not completely surprising given that the immunity protein has an incredibly high affinity for the activity protein and that pLac is known to be leaky. Even a small concentration of immunity protein could protect un-induced cells against the DNase activity of the colicin activity protein.<br />
<br />
===Quantitative Characterization of the Antitoxin Plasmid===<br />
<br />
We characterized the plasmid pLac + ColE2 immunity gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]) by testing the response of the cells when we mix them with colicin producing cells in various concentration with also various concentration of IPTG as the inducer of pLac. We counted the number of surviving colonies to see the effectiveness of the immunity protein in protecting the cells from the colicin activity protein. <br />
<br />
====Experimental Setup====<br />
'''Cell types'''<br />
*Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2] <br />
*NEB.CmR: NEB turbo cells transformed with [http://partsregistry.org/Part:pSB3C5 pSB3C5]).<br />
*NEB.Imm: NEB turbo cells transformed with the antitoxin plasmid ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K914001 BBa_K914001] inside [http://partsregistry.org/Part:pSB3C5 pSB3C5]).<br />
<br />
'''Protocol'''<br />
#Grow overnight cultures of NEB.CmR, NEB.Imm each with antibiotic (Cm) and Col E2.<br />
#Centrifuge and resuspend in LB.<br />
#Prepare 5 tubes of 10ml LB and put 40uL of NEB.Imm cells into 4 tubes and NEB.CmR cells in 1 tube.<br />
#Incubate 37C until the OD reaches 0.2.<br />
#Put IPTG with different concentration in 3 NEB.Imm tubes (0.1mM, 0.05mM, 0.025mM).<br />
#Incubate again until the OD reaches 1.0.<br />
#Take each 0.5ml and mix with 0.5ml Colicin cells (saturated, saturated diluted 1000x, plain LB with corresponding amount of IPTG) in small 2ml eppendorf tubes.<br />
#Incubate for 30 minutes.<br />
#Dilute 10000x (100x then 100x) in MgSO4 e-2M.<br />
#Plate 20uL in Cm to kill the Colicin cells and leave the immune cells.<br />
Note: 1ml of cells of OD 1.0 has 1e9 cells, so we expect if all cells survive we will get 1000 colonies at the end.<br />
<br />
====Results====<br />
<!--<br />
The table shows the number of colonies survive in each plate IPTG x Colicin. <br />
<center><br />
{| style="border-spacing:0;"<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| <br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 100uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 10uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1uM IPTG<br />
| style="border-top:0.035cm solid #000000;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 0 IPTG<br />
| style="border:0.035cm solid #000000;padding:0.097cm;"| pSB3C5 (-) control<br />
<br />
|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1x Colicin<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 94<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 79<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 73<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 42<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 0<br />
<br />
|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 1000x Colicin<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 9<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 24<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 26<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 74<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 0<br />
<br />
|-<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| No Colicin (+) control<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 36<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 28<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 30<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:none;padding:0.097cm;"| 18<br />
| style="border-top:none;border-bottom:0.035cm solid #000000;border-left:0.035cm solid #000000;border-right:0.035cm solid #000000;padding:0.097cm;"| 27<br />
<br />
|}<br />
<br />
--><br />
<center><br />
[[Image:Colicin-assay.png |600px]] </center><br />
<br />
<br />
In this experiment we expected to see more survival if we induce the cells with more IPTG and/or mix them with less Colicin cells. Given that from the previous experiment the immunity protein turned out to be independent to IPTG induction, we treated all the NEB.Imm data as the same. The results showed that the immunity protein can indeed protect the cells from saturated colicin. If we do not put the Colicin cells in the mixture, the numbers of survival cells of NEB.Imm (BBa_K914001) and NEB.CmR (pSB3C5) are almost the same. But if we put the Colicin cells, even very diluted, the NEB.CmR cells could not survive.<br />
<br />
We noticed that the survival on the positive controls (mixture without Colicin) was somehow less than the one with Colicin cells. We guessed that the problems are<br />
*The plates have more cells when they have more Colicin: the Colicin cells maybe took the Immunity/Cm-resistance plasmid<br />
*Less cells on the plate without Colicin (and very few cells overall compared to the theoretical calculation): the cells may have died because they loose the Immunity/Cm-resistance plasmid during the incubation without antibiotic (which we did to avoid killing the Colicin cells).<br />
Therefore we still need to improve protocol to provide a more reliable quantitative data.<br />
<br />
<div id="boston"><br />
===Testing the effects of colicin-containing protein extract on E.coli===<br />
<br />
We mix Z1 cells with the supernatant of centrifuged Colicin cells (exctract with the ColE2 protein). Therefore we can put the antibiotic during the whole experiment to avoid plasmid loss.<br />
<br />
====Experimental Setup====<br />
'''Cell types'''<br />
*colicin-producing cells<br />
*Z1 cells with immunity<br />
*Z1 cells without immunity as a control<br />
<br />
'''Protocol'''<br />
#Grow overnight cultures of colicin-producing cells, Z1 cells with immunity and Z1 cells without immunity.<br />
#For the colicin-producing cells (protocol adapted from http://www.piercenet.com/browse.cfm?fldID=06010401 ):<br />
## Centrifuge at 5000g for 10 mins<br />
## Weigh the falcons with the cell pellet and add 4mL of B-PER Reagent per gram of cell pellet. Pipette the suspension up and down until it is homogeneous<br />
##Incubate 10-15 minutes at room temperature<br />
## Pour the lysate into eppendorff tubes and centrifuge it at 15,000 × g for 5 minutes to separate soluble proteins from the insoluble proteins. Keep the soluble fraction: this is the protein extract containing colicins.<br />
<br />
#For the immune and non-immune cells:<br />
## Dilute the overnight cultures 1/100 into 10-ml cultures (with antibiotics):<br />
### 3 x Z1 cells without immunity<br />
### 3 x Z1 cells with immunity, induced with IPTG<br />
### 3 x Z1 cells with immunity, non-induced<br />
## Grow the cultures for at least 1h, make sure that the OD is more or less the same for all cultures (tip: you can keep a culture with a higher OD at 4°C to wait until the other cultures reach the same OD)<br />
## Mix the immune/non-immune cells with the colicin extract, in different proportions (cells/colicin extract 9:1 and 5:5). For controls, mix immune/non-immune cells with the B-PER buffer, in same proportions<br />
# Put the cells into a 96 well plate for Tecan, 2 wells per each of the cultures<br />
# Put the plate into Tecan, and make OD measurements each 10mins<br />
<br />
====Results====<br />
[[Image:PB2012_Immunity_exp.jpeg|700px|center]]<br />
Growth curves of cells with or without protein extract of colicin producing cells.<br />
We compare the growth of cells that have immunity protein or not. Colicin-containing protein extract significantly impedes the growth of cells that do not have immunity (green curve), whereas it does not affect cells expressing immunity (blue curve), and they grow as well as positive controls (red and purple lines, respectively).<br />
Remarks: Immune cells grow even better in the presence of the colicin extract than in the presence of the buffer. This can be explained by the fact that the buffer used for protein extraction leads to cell lysis. In future, we will use the extract of cells that do not produce colicin, as a positive control. Also, in this setup, the step-like trend of the growth curve for the sensitive cells in the presence of colicin extract (blue) can be explained by the degradation of the colicin, and/or by the fact that it all colicin present could be used up at some point, and not affect further growth of cells. <br />
In conclusion, these results show that colicin produced by cells prevents the growth or even kills sensitive cells, whereas the cells expressing the immunity protein survive.<br />
<br />
</div><br />
<br />
====Persperctives====<br />
We suggest in the future we could give stronger stress to the Colicin to induce more the SOS promoter; such as UV light.<br />
<br />
==Discussion==<br />
Cloning the colicin E2 activity protein, given its lethal nature, is a great challenge for us. We took several approaches. We established immune strains expressing excess immunity protein in which we could transform the toxin plasmid. We tried cloning the toxin without a promoter and finally tried various vectors with and with out promoters. Although the immune strains are viable, transformable, and prove immune to wild type colicin E2 cells, we have yet to successfully transform a single toxin plasmid. <br />
<br />
However, developing and characterizing the immune strains was not as straight forward as we imagined. Whether in NEB turbo or MG1655 Z1 cells, the immune strains grew incredibly slowly. Despite being under the control of pLac, their immunity proved somewhat independent of IPTG, as both un-induced and induced cells prove immune. In addition, we had some strange toxicity assay results, partially resolved by the explanation of colicin E2 contamination. <br />
<br />
To see more details on the toxicity assays, click [http://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Notebooks/suicide_group/day_by_day//2012/09/08 here]<br />
<br />
'''UPDATE:'''<br />
We are still struggling with cloning the colicin E2 activity protein, however we have managed to clone the coding sequence and have some more promising results using the sRNA approach described [https://2012.igem.org/Team:Paris_Bettencourt/Delay here]<br />
<br />
==Related Parts and Links==<br />
*[http://partsregistry.org/Part:BBa_K131000 BBa_K131000] Colicin E2 operon, designed by Kevin McLeod, group: iGEM08_Calgary_Wetware (2008-07-22)<br />
*[http://partsregistry.org/Part:BBa_R0011 BBa_R0011] Promoter (lacI regulated, lambda pL hybrid), designed by Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton, group: Registry (2003-01-31)<br />
*[http://partsregistry.org/Part:BBa_R0011:Experience/iGEM10_Kyoto BBa_R0011:Experience/iGEM10 Kyoto] pLac model<br />
*[http://partsregistry.org/pSB4K5 pSB4K5 ] Low copy plasmid containing kanamycin resistance<br />
*[http://partsregistry.org/pSB3C5 pSB3C5 ] Low-medium copy plasmid containing chloramphenicol resistance<br />
*[http://partsregistry.org/wiki/index.php/Part:BBa_J23108 BBa_J23108] Anderson Constitutive Promoter with relative strength 0.51 (RFP reporter)<br />
*[http://partsregistry.org/wiki/index.php/Part:BBa_J23102 BBa_J23102] Anderson Constitutive Promoter with relative strength 0.86 (RFP reporter)<br />
*[http://partsregistry.org/Part:BBa_K914016 K914016] coding sequence of Colicin E2<br />
<br />
==References==<br />
#Cascales E, et al. (2007) Colicin biology. Microbiol Mol Biol Rev 71:158–229. <br />
#Majeed G, Gillor O, Kerr B, Riley MA. (2011) Competitive interactions in Escherichia coli populations: the role of bacteriocins. The ISME Journal 5, 71-81.<br />
#Pugsley AP. (1985) Escherichia coli K12 strains for use in the identification and characterication of colicins. J Gen Microbiol 131: 369-376.<br />
#Kleanthous C. (2010) Swimming against the tide: progress and challenges in our understanding of colicin translocation. Nat Microbiol Rev 8:843-848.<br />
#Torres B, et al. (2003) A dual lethal system to enhance containment of recombinant micr-organisms. Microbiol 149: 3595-3601.<br />
<br />
==Appendix==<br />
===ColicinE2 Plasmid Map===<br />
[[File:ColE2P9.jpg|frameless|center|500px]]<br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Semantic_containmentTeam:Paris Bettencourt/Semantic containment2012-10-27T00:48:14Z<p>Aleksandra: /* Conclusion & Perspectives */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<div id="grouptitle">Semantic containment</div><br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Aims'''<br />
* Creating a semantic containment system to prevent gene expression in natural organisms<br />
* Characterize the system<br />
* Use this system in all genes of the system, the critical genes first (e.g. [https://2012.igem.org/Team:Paris_Bettencourt/Suicide colicin])<br />
<br />
'''System'''<br />
*An amber codon (stop codon) embedded in protein genes to prevent their expression and an amber suppressor system in our genetically engineered bacteria<br />
<br />
'''Achievements :'''<br />
* Construction and characterization of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914000 K914000] : P<sub>Lac</sub>-supD-T : tRNA amber suppressor<br />
** [http://partsregistry.org/Part:BBa_K914009 K914009] : P1003* Ser133->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
<br />
Both part were well [[#Results|characterized]] and works well. For the second parts, we show that as expected, one mutation is quite leaky, although it works qualitatively, but one mutation is not enough if we want to release such parts in nature. Other reasons emphasize this observation, notably the weakness of being at one mutation to recover the protein functionality.<br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety Semantic containment]. The aim of this category is to let people improving each part by adding for instance other amber mutations to existing part to increase the containment.<br />
<div id="boston"><br />
'''Achievements : '''<br />
* Construction and characterization of 1 biobrick :<br />
** [http://partsregistry.org/Part:BBa_K914018 K914018] : P1003** Ser133 & Ser203 ->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
* Construction of 1 plasmid backbone : <br />
** [http://partsregistry.org/Part:BBa_K914012 K914012] : pSB1A2 with one Amber Codon : Ampicillin gene resistance with 1 amber mutation<br />
</div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
==Overview==<br />
We want to prevent our genetic construct from conferring an advantage to other organisms or alter them phenotypically. Minimizing horizontal gene transfer (HGT), either by conjugation, transduction, or transformation is thus our main concern. As these processes involve two parties, the genetically modified bacteria and some wild type population partners, and as we will not modify wild type populations, we cannot assume that HGT is fully avoidable. Semantic containment<sup>[</sup><sup>[[#References|1]]</sup><sup>]</sup> means that our bacteria won't be able to "speak" with other organisms, since they don't speak the same "language", the language being DNA. Our system will read the stop codon TAG as the amino-acid serine. It means that in our bacteria the stop codon will be translated into a serine, whereas in wild type bacteria this protein will be truncated and will not confer an advantage to these cells. The 'TAG' codon has been chosen because of its low frequency in ''E. coli'' genome (314 occurrences), and also because for further applications, Church Lab tries to remove all amber codon of an ''E. coli'' strain<sup>[</sup><sup>[[#References|2]]</sup><sup>]</sup>. Although it has been demonstrate that the over-expression of a tRNA amber suppressor sole does not affect its growth rate nor the morphology of ''E. coli''<sup>[</sup><sup>[[#References|3]]</sup><sup>]</sup>.<br />
[[File:ConceptSC.png|thumb|center|800px|'''Figure 1 :''' Semantic containment principle. The information carried by the DNA cannot be read by other organism]]<br />
<br />
==Objectives==<br />
We want to create a new way to contain semantically genes, by replacing an amino-acid codon by a stop codon (amber codon, 'TAG'), and in our synthetic cell this stop codon will be read as the amino-acid. Here we want to show that this system works as expected. First we had to choose between two tRNA amber suppressor, either serine, or tyrosine that are available in the part registry. For that we calculate the abilities for the amber codon to reverse to a serine or tyrosine or related amino-acid that could conserve the function. Secondly we create a biobrick with a tRNA Amber suppressor ([http://partsregistry.org/Part:BBa_K914000 BBa_K914000]), in order to have a reliable biobrick, with characterization of it. Thirdly, to test the latter biobrick, we built a biobrick which is a kanamycin resistance gene (P1003) with one amber mutation added instead of the serine 133 ([http://partsregistry.org/Part:BBa_K914009 BBa_K914009]).<br />
<br />
K914000 is the construction P<sub>Lac</sub>-supD-T, and is named supD in the rest of the page. K914009 is the P1003 gene (kanamycin resistance gene) with the serine 133 which is replaced by a amber codon 'TAG'. Its name is Kan<sup>*</sup>.<br />
<br />
With more time we will try to increase the robustness of this system, which is null when the tRNA amber suppressor is transferred too. We will try to create a new library of plasmid backbones in the part registry, where all backbones have at least two amber mutations. The idea is that all the community will be able to improve this library, either by adding new contained backbones, or by adding amber mutations on the same backbone, or add semantic containment to any other gene.<br />
<br />
==Design==<br />
===How to choose between serine and tyrosine ? ===<br />
At one mutation of the 'TAG' codon we can have one serine or two tyrosine (among others). Even if the serine seems more interesting by this simple observation, we still want to know which of these two amino-acids is the less robust to mutation from a 'TAG' codon (amber codon) including similarity property of amino-acids. We calculated a score of weakness. The weakness of an amino-acid is defined here by its abilities to not revert to the same amino-acid or any other similar, from the amber codon.<br />
The score is calculated using the following formula :<br />
<center><br />
<html><br />
<img src="http://www.openwetware.org/images/math/6/7/3/673fca1559040fa2561513142404b963.png"><br />
</html><br />
</center><br />
<br />
Where i is one of the nine amino-acids accessible after 1 mutation. Subst(AA,AAi) calculate the similarity score, using a BLOSUM100 matrix, between serine or tyrosine (AA) and one of the nine amino-acids around (AAi). The lowest score is the weakest. The BLOSUM100 (BLOck SUbstitution Matrix) is constructed using local alignment of sequenced less than 100% identical<sup>[</sup><sup>[[#References|4,5]]</sup><sup>]</sup>, and is consequently adapted to appreciate the effect of a single mutation.<br />
<br />
===Weakness calculation===<br />
<br />
We wrote a Python script that can calculate the score of weakness, with all amino-acids (not only serine or tyrosine), and will sort a list of score. Data shown in [[#Other_amino-acids_weakness_score|appendix]] for other amino-acids.<br />
<br />
The scores are, with a mutation rate of 10<sup>-9</sup> for the serine and tyrosine:<br />
<br />
*BLOSUM100 :<br />
** Serine : Score<sup>S</sup><sub>W</sub> = -3.33e-09<br />
** Tyrosine : Score<sup>Y</sup><sub>W</sub> = -1.00e-09<br />
<br />
The amber codon is less likely to revert into a serine or similar.<br />
<br />
Also, we favored serine replacement over tyrosine, because the frequency of serine in the ''E. coli'' genome is superior to the tyrosine one. It turns out that S has 57,88 codons over 1000 codons when Y has 28,59 codons over 1000 codons ([http://openwetware.org/wiki/Escherichia_coli/Codon_usage Codon usage]). Therefor, it might be more convenient to replace a serine than a tyrosine, because proteins would more likely to contain many serines than tyrosines.<br />
<br />
===Can the tRNA rescues the Kan<sup>R</sup> phenotype ? (Qualitative experiment)===<br />
[[File:SCsupD.gif|thumb|center|800px|'''Figure 3 :''' Our hypothesis : supD can rescue the Kan<sup>R</sup> phenotype. '''A''' production of the mRNA from the Kan* gene. '''B''' The mRNA is translated only if there is a tRNA amber suppressor (supD)]]<br />
<br />
To do so, we will transform a plasmid with a the Kan<sup>*</sup> gene into a MG1655 strain that contain either pSB1C3::supD, or pSB1C3::RFP. We plate them on Chloramphenicol and Kanamycin. The Kan<sup>*</sup> is supposed to be non functional without an amber suppressor.<br />
<br />
===Is it working well? Is the amber mutation leaky? (Quantitative experiment)===<br />
[[File:Design1SC.png|thumb|right|600px| '''Figure 2 :''' Schema of the four strains used for the quantitative experiments]]<br />
<br><br />
<br><br />
We used the following strains, all in the ''E. coli'' strain MG1655. To quantify the leakiness in terms of expression of the Kan<sup>*</sup> gene, we performed real time experiment, where we measured the growth rate (through OD<sub>600</sub> measurement) of each of these strains in different concentrations of kanamycin : <br />
<br />
(1) '''pSB1A1::Kan<sup>R</sup> + pSB1C3::supD''':<br> positive control : Expresses constitutively kanamycine resistance gene.<br />
<br />
(2) '''pSB1A1::Kan<sup>*</sup> + pSB1C3::supD''':<br> construction : Is supD as efficient as the positive control?<br />
<br />
(3) '''pSB1A1::Kan<sup>*</sup> + pSB1C3::RFP''':<br> construction : Is Kan* as unefficient as the negative control?<br />
<br />
(4) '''pSB1A2::P<sub>Lac</sub> + pSB1C3::supD''':<br> negative control : No kanamycin resistance gene.<br />
<br />
In case of leakiness, the Kan* + RFP strain (3) will be able to grow in higher concentration of kanamycin than the negative control (4).<br />
<br><br />
<br><br />
<br />
==Experiments and results==<br />
<br />
<br />
===Qualitative characterization of K914000 (supD) and K914009 (Kan*)===<br />
[[File:GraphCFUqual.png|thumb|border=0|500px|right|'''Figure 3 :''' Number of CFU/µg of plasmid after the different concentration]]<br />
<br />
<br />
After preparing electro-competent M1655 cells with either pSB1C3::supD or pSB1C3::RFP. We transform the plasmid pSB1A1::Kan<sup>*</sup> in both competent cells. After transformation, cells are plated on Cm+Kan.<br />
<br />
We can observe that without any plasmids transformed no cells grow, or when we transform another plasmid with no Kan resistance gene, but with an Amp gene resistance (and plated on Cm+Amp), colonies appear in the strain with RFP, unfortunately the other control (with supD) did not work this time, hence there is no picture of it. But for the quantitative experiment we need that control too (4), and we manage to do it that times. But the pSB1A1::Kan<sup>*</sup> can express the kanamycin resistance phenotype only in the strain containing the supD gene.<br />
<br />
We can conclude here that the supD gene can rescue the phenotype Kan<sup>S</sup> by allowing the correct expression of the kan gene P1003.<br />
<br />
<center><br />
<gallery caption="plates of the double transformation" perrow="5"><br />
File:RFP_pur_Cm_Kan.png|MG1655 + pSB1C3::supD only Selection : Cm+Kan<br />
File:RFP_pur_Cm_Kan.jpg|MG1655 + pSB1C3::RFP only Selection : Cm+Kan<br />
File:SupD%2BKan*_pur_Cm_Kan.jpg|MG1655 + pSB1C3::supD + pSB1A1::Kan<sup>*</sup> Selection : Cm+Kan<br />
File:RFP%2BKan*_pur_Cm_Kan.jpg|MG1655 + pSB1C3::RFP + pSB1A1::Kan<sup>*</sup> Selection : Cm+Kan<br />
File:RFP%2BpLac_200uL_Cm_Amp.jpg|MG1655 + pSB1C3::RFP + pSB1A2::pLac<sup>*</sup> Selection : Cm+Amp<br />
</gallery><br />
</center><br />
<br />
===Quantitative characterization of K914000 (supD) and K914009 (Kan*)===<br />
Here we characterize both biobrick quantitatively. First, we are going to confirm the qualitative result for the part K914000 and then we will determine how a single amino-acid substitution is leaky.<br />
====Experimental setup====<br />
Once the construction is made (Fig. 2), we double transformed the plasmids into MG1655 strains, that does not contain any amber suppressor. We will work with three replicates of each strains, and for each strain we will be in 8 different conditions of antibiotic resistance. The antibiotic used is kanamycin, an aminoglycoside interfering with the translation. The range of concentration goes from 4 times as much as the usual concentration (100 µg/mL) to 8 times less. The 96 wells plate is then incubated in a plate reader that take measurements of OD<sub>600</sub> every 6 minutes. This measure is correlated to the number of cell in the well. <br />
Each well contains 200µL of LB (Lysogeny broth, aka Luria Bertani), chloramphenicol and ampicilin at their usual concentration, the dilution of cells and different amount of kanamycin. An overlay of 50µL of mineral oil is added on the top. The measurement lasted approximately 16 hours and 30 min.<br />
[[File:SCquantitativec.png|thumb|center|800px|'''Figure 4 :''' The over night (O/N) culture is diluted twice, first to normalize all samples, and then to start with low concentration of cells.]]<br />
<br />
====Results====<br />
<br />
First we observed that the qualitative result is reproduce here, the supD gene rescues the kanamycin resistance at any concentration of the antibiotic. It shows also that there is no disadvantage to use the supD amber suppressor compared to the wild type kanamycin gene resistance, since there is no difference of growth rate (Figure 4B and 4C). However the leakiness of the Kan* gene is higher than expected, and thus one mutation is not sufficient for the containment. Indeed, at usual concentration, Kan* gene manages to express an antibiotic resistance, even though lower than Kan<sup>R</sup> or supD (Figure 4A). Here we define the growth rate as the OD<sub>600</sub> observed at a given time (here t=8h20'), in order to overcome the fact that Kan*+RFP does not have a clear exponential phase. The fact that the strain (3) grows faster and can have higher OD<sub>600</sub> may be explain by the fact that supD is deleterious for the strain, by removing some stop codon of other genes for instance. <br />
<br />
[[File:SCquantitativeF1a.png|thumb|center|800px|'''Figure 4A and 4B:''' The bar graph represent the OD<sub>600</sub> at time = 8h20' (black line Figure 4C) for the different strains at a given kanamycin concentration (black line on Figure 4B). On B, we observe the variations of the OD<sub>600</sub> at different kanamycin concentrations, at the same time.]]<br />
<br />
[[File:SCquantitativeF2.png|thumb|center|800px|'''Figure 4C:''' The variations of the OD<sub>600</sub> is observed in function of the time, for different concentrations of kanamycin (400µg/mL, 100µg/mL, 25µg/mL).]]<br />
<br />
<div id="boston"><br />
===Quantitative characterization of K914018 (Kan**)===<br />
Here we perform the exact same experiment than the previous one, except that kanamycin resistant gene is contained with two amber mutations instead of one. It is observed that the positive and negative controls behave correctly, and similarly to the previous experiment, it is also the case for the complementation of Kan** with supD. That means that two mutation are not a problem for the cell. However, we notice that there is no more leakiness in the system at usual concentration, even at low concentration. Actually the double kanamycin resistant gene with two mutation behave exactly like the negative control (Plac + supD) which means that semantic containment is working well.<br />
<br />
[[File:SCquantitative**2.png|thumb|center|800px|'''Figure 5A and 5B:''' The bar graph represent the OD<sub>600</sub> at time = 8h37' (black line Figure 5C) for the different strains at a given kanamycin concentration (black line on Figure 5B). On B, we observe the variations of the OD<sub>600</sub> at different kanamycin concentrations, at the same moment.]]<br />
<br />
[[File:SCquantitative**1.png|thumb|center|801px|'''Figure 5C:''' The variations of the OD<sub>600</sub> is observed in function of the time, for different concentrations of kanamycin (400µg/mL, 100µg/mL, 25µg/mL and 3.125µg/mL).]]<br />
<br />
=== Comparison between one and two mutation ===<br />
Here is a graph that gather both results, to highlight the effect of the second mutation. The results are normalized intra group (experiment with the one-mutated gene, and with the two-mutated gene) in order to have the same scale to compare them. We took the average of the positive (KanR+supD) and the negative (Plac+supD) controls. <br />
[[File:SCquantitative**bilan.png|thumb|center|800px|'''Figure 6:''' Variation of OD<sub>600</sub> in different concentrations of [Kan] (µg/mL) at t= 8,37h]]<br />
<br />
</div><br />
<br />
==Conclusion & Perspectives==<br />
<br />
This work demonstrates that semantic containment can be achieved by changing an amino-acid into an amber codon. We saw that one codon replacement is not enough because of some leakiness, and demonstrated that upon the replacement of two codons, the system isn't leaky at all. It means that for a robust semantic containment system we need 3 codon replacements, because with two replacements we are only one mutation away of a leaky expression. The underlying idea is ''in fine'' to create a library of semantic-contained genes and backbones that would be improved by anybody, by either adding new semantic systems, or increasing the degree of containment by adding mutations on already existing parts. This library is already started with the previously described part and with a plasmid backbone that has only one mutation so far. But we also showed that tRNA amber suppressor might be deleterious for the cell. This trade-off between the leakiness and the toxicity has to be studied in order to optimize the system.<br />
<br />
Further experiments should improve the robustness of this system by adding a new security component that would ensure that as much as three genes would have to be transferred by HGT, to express synthetic gene in natural bacteria, thus cousing our containment system to fail. In our current design, the system fails if the tRNA amber suppressor gene is transferred with another semantic gene into a natural bacteria. The idea is to add a semantic containment for the tRNA. Since it is a tRNA, we cannot replace amino-acids because it is not translated. In order to fix that problem we will construct the following system. The tRNA supD gene will be under a T7 promoter which is orthogonal, meaning that it needs a special RNA polymerase (T7 RNA polymerase) to transcribe the gene (here it would be supD), but we would mutate the T7 RNA polymerase with several amber mutations. It means that we would need to transfer these two genes with the semantically contained gene in order to have something functional in the other organism, which is very unlikely to happen. Further experiments will elucidate the probability of such an event. A scheme of this system is depicted in Figure 5.<br />
This system needs to be activated in the lab, for example by transforming a plasmid with the wild type T7 RNA polymerase gene with another antibiotic gene resistance, say X. Then we would remove antibiotic X and loose the plasmid with wild type T7 polymerase when the positive feedback loop starts. We could also activate the system by transforming a wild-type T7 RNA polymerase carrying plasmid with conditional origin of replication, such as temperature-sensitive plasmids.<br />
<br />
[[File:SCT7supD.png|thumb|800px|center|'''Figure 5 : '''Once the mRNA of the T7 RNA polymerase is transcribed (A), it needs the tRNA amber suppressor (B) to let the ribosome translate the mRNA into a functional protein. Then the T7 RNA polymerase will be able to transcribe the supD gene into this tRNA amber suppressor (C). Then it is a positive feedback loop, we need to start this system by adding either tRNA in the medium or T7 RNA pol wild type gene.]]<br />
<br />
==References & Appendix==<br />
===References===<br />
[[#Overview|1]] - Marliere, P. The farther, the safer : a manifesto for securely navigating synthetic species away from the old living world. System and Synthetic Biology 3, 77-84 (2009). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759432/ Paper]<br />
<br />
[[#Overview|2]] - Isaacs, F.J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science (New York, N.Y.) 333, 348-53 (2011). [http://arep.med.harvard.edu/pdf/Isaacs_Sci_11.pdf Paper]<br />
<br />
[[#Overview|3]] - Anderson, J.C., Voigt, C. a & Arkin, A.P. Environmental signal integration by a modular AND gate. Molecular systems biology 3, 133 (2007). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1964800/ Paper]<br />
<br />
[[#Calculation_of_serine_and_tyrosine_weakness|4]] - S Henikoff and J G Henikoff. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A. 1992 November 15; 89(22): 10915–10919. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC50453/pdf/pnas01096-0363.pdf Paper]<br />
<br />
[[#Calculation_of_serine_and_tyrosine_weakness|5]] - Jorja G. Henikoff, Steven Henikoff, Blocks database and its applications, Methods in Enzymology, Academic Press, 266, 88-105 (1996). [http://www.sciencedirect.com/science/article/pii/S007668799666008X Paper]<br />
<br />
===Appendix===<br />
====Genotype of strain used====<br />
<br />
* MG1655 : F- λ- ilvG- rfb-50 rph-1 <br />
<br />
==== Other amino-acids weakness score ====<br />
<br />
* Weakness score with a BLOSUM 100, mutation rate = 10<sup>-9</sup>, initial Codon : TAG<br />
<br />
[[#Weakness_calculation|main result part]]<br />
<br />
('A', [-4.000000002555556e-09])<br />
<br />
('C', [-6.777777782148149e-09])<br />
<br />
('D', [-5.2222222257407416e-09])<br />
<br />
('E', [-3.222222225407407e-09])<br />
<br />
('F', [-3.0000000040740746e-09])<br />
<br />
('G', [-6.555555560444445e-09])<br />
<br />
('H', [-2.888888891555555e-09])<br />
<br />
('I', [-5.111111115777778e-09])<br />
<br />
('K', [-2.6666666694074067e-09])<br />
<br />
('L', [-4.333333337555555e-09])<br />
<br />
('M', [-4.000000003185186e-09])<br />
<br />
('N', [-4.3333333354074076e-09])<br />
<br />
('P', [-5.888888893518519e-09])<br />
<br />
('Q', [-1.6666666689629633e-09])<br />
<br />
('R', [-3.888888891888888e-09])<br />
<br />
('S', [-3.333333334925926e-09])<br />
<br />
('T', [-4.22222222462963e-09])<br />
<br />
('V', [-4.888888893111112e-09])<br />
<br />
('W', [-2.4444444508518513e-09])<br />
<br />
('Y', [-1.0000000044444444e-09])<br />
<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Semantic_containmentTeam:Paris Bettencourt/Semantic containment2012-10-27T00:35:29Z<p>Aleksandra: /* Conclusion & Perspectives */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<div id="grouptitle">Semantic containment</div><br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Aims'''<br />
* Creating a semantic containment system to prevent gene expression in natural organisms<br />
* Characterize the system<br />
* Use this system in all genes of the system, the critical genes first (e.g. [https://2012.igem.org/Team:Paris_Bettencourt/Suicide colicin])<br />
<br />
'''System'''<br />
*An amber codon (stop codon) embedded in protein genes to prevent their expression and an amber suppressor system in our genetically engineered bacteria<br />
<br />
'''Achievements :'''<br />
* Construction and characterization of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914000 K914000] : P<sub>Lac</sub>-supD-T : tRNA amber suppressor<br />
** [http://partsregistry.org/Part:BBa_K914009 K914009] : P1003* Ser133->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
<br />
Both part were well [[#Results|characterized]] and works well. For the second parts, we show that as expected, one mutation is quite leaky, although it works qualitatively, but one mutation is not enough if we want to release such parts in nature. Other reasons emphasize this observation, notably the weakness of being at one mutation to recover the protein functionality.<br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety Semantic containment]. The aim of this category is to let people improving each part by adding for instance other amber mutations to existing part to increase the containment.<br />
<div id="boston"><br />
'''Achievements : '''<br />
* Construction and characterization of 1 biobrick :<br />
** [http://partsregistry.org/Part:BBa_K914018 K914018] : P1003** Ser133 & Ser203 ->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
* Construction of 1 plasmid backbone : <br />
** [http://partsregistry.org/Part:BBa_K914012 K914012] : pSB1A2 with one Amber Codon : Ampicillin gene resistance with 1 amber mutation<br />
</div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
==Overview==<br />
We want to prevent our genetic construct from conferring an advantage to other organisms or alter them phenotypically. Minimizing horizontal gene transfer (HGT), either by conjugation, transduction, or transformation is thus our main concern. As these processes involve two parties, the genetically modified bacteria and some wild type population partners, and as we will not modify wild type populations, we cannot assume that HGT is fully avoidable. Semantic containment<sup>[</sup><sup>[[#References|1]]</sup><sup>]</sup> means that our bacteria won't be able to "speak" with other organisms, since they don't speak the same "language", the language being DNA. Our system will read the stop codon TAG as the amino-acid serine. It means that in our bacteria the stop codon will be translated into a serine, whereas in wild type bacteria this protein will be truncated and will not confer an advantage to these cells. The 'TAG' codon has been chosen because of its low frequency in ''E. coli'' genome (314 occurrences), and also because for further applications, Church Lab tries to remove all amber codon of an ''E. coli'' strain<sup>[</sup><sup>[[#References|2]]</sup><sup>]</sup>. Although it has been demonstrate that the over-expression of a tRNA amber suppressor sole does not affect its growth rate nor the morphology of ''E. coli''<sup>[</sup><sup>[[#References|3]]</sup><sup>]</sup>.<br />
[[File:ConceptSC.png|thumb|center|800px|'''Figure 1 :''' Semantic containment principle. The information carried by the DNA cannot be read by other organism]]<br />
<br />
==Objectives==<br />
We want to create a new way to contain semantically genes, by replacing an amino-acid codon by a stop codon (amber codon, 'TAG'), and in our synthetic cell this stop codon will be read as the amino-acid. Here we want to show that this system works as expected. First we had to choose between two tRNA amber suppressor, either serine, or tyrosine that are available in the part registry. For that we calculate the abilities for the amber codon to reverse to a serine or tyrosine or related amino-acid that could conserve the function. Secondly we create a biobrick with a tRNA Amber suppressor ([http://partsregistry.org/Part:BBa_K914000 BBa_K914000]), in order to have a reliable biobrick, with characterization of it. Thirdly, to test the latter biobrick, we built a biobrick which is a kanamycin resistance gene (P1003) with one amber mutation added instead of the serine 133 ([http://partsregistry.org/Part:BBa_K914009 BBa_K914009]).<br />
<br />
K914000 is the construction P<sub>Lac</sub>-supD-T, and is named supD in the rest of the page. K914009 is the P1003 gene (kanamycin resistance gene) with the serine 133 which is replaced by a amber codon 'TAG'. Its name is Kan<sup>*</sup>.<br />
<br />
With more time we will try to increase the robustness of this system, which is null when the tRNA amber suppressor is transferred too. We will try to create a new library of plasmid backbones in the part registry, where all backbones have at least two amber mutations. The idea is that all the community will be able to improve this library, either by adding new contained backbones, or by adding amber mutations on the same backbone, or add semantic containment to any other gene.<br />
<br />
==Design==<br />
===How to choose between serine and tyrosine ? ===<br />
At one mutation of the 'TAG' codon we can have one serine or two tyrosine (among others). Even if the serine seems more interesting by this simple observation, we still want to know which of these two amino-acids is the less robust to mutation from a 'TAG' codon (amber codon) including similarity property of amino-acids. We calculated a score of weakness. The weakness of an amino-acid is defined here by its abilities to not revert to the same amino-acid or any other similar, from the amber codon.<br />
The score is calculated using the following formula :<br />
<center><br />
<html><br />
<img src="http://www.openwetware.org/images/math/6/7/3/673fca1559040fa2561513142404b963.png"><br />
</html><br />
</center><br />
<br />
Where i is one of the nine amino-acids accessible after 1 mutation. Subst(AA,AAi) calculate the similarity score, using a BLOSUM100 matrix, between serine or tyrosine (AA) and one of the nine amino-acids around (AAi). The lowest score is the weakest. The BLOSUM100 (BLOck SUbstitution Matrix) is constructed using local alignment of sequenced less than 100% identical<sup>[</sup><sup>[[#References|4,5]]</sup><sup>]</sup>, and is consequently adapted to appreciate the effect of a single mutation.<br />
<br />
===Weakness calculation===<br />
<br />
We wrote a Python script that can calculate the score of weakness, with all amino-acids (not only serine or tyrosine), and will sort a list of score. Data shown in [[#Other_amino-acids_weakness_score|appendix]] for other amino-acids.<br />
<br />
The scores are, with a mutation rate of 10<sup>-9</sup> for the serine and tyrosine:<br />
<br />
*BLOSUM100 :<br />
** Serine : Score<sup>S</sup><sub>W</sub> = -3.33e-09<br />
** Tyrosine : Score<sup>Y</sup><sub>W</sub> = -1.00e-09<br />
<br />
The amber codon is less likely to revert into a serine or similar.<br />
<br />
Also, we favored serine replacement over tyrosine, because the frequency of serine in the ''E. coli'' genome is superior to the tyrosine one. It turns out that S has 57,88 codons over 1000 codons when Y has 28,59 codons over 1000 codons ([http://openwetware.org/wiki/Escherichia_coli/Codon_usage Codon usage]). Therefor, it might be more convenient to replace a serine than a tyrosine, because proteins would more likely to contain many serines than tyrosines.<br />
<br />
===Can the tRNA rescues the Kan<sup>R</sup> phenotype ? (Qualitative experiment)===<br />
[[File:SCsupD.gif|thumb|center|800px|'''Figure 3 :''' Our hypothesis : supD can rescue the Kan<sup>R</sup> phenotype. '''A''' production of the mRNA from the Kan* gene. '''B''' The mRNA is translated only if there is a tRNA amber suppressor (supD)]]<br />
<br />
To do so, we will transform a plasmid with a the Kan<sup>*</sup> gene into a MG1655 strain that contain either pSB1C3::supD, or pSB1C3::RFP. We plate them on Chloramphenicol and Kanamycin. The Kan<sup>*</sup> is supposed to be non functional without an amber suppressor.<br />
<br />
===Is it working well? Is the amber mutation leaky? (Quantitative experiment)===<br />
[[File:Design1SC.png|thumb|right|600px| '''Figure 2 :''' Schema of the four strains used for the quantitative experiments]]<br />
<br><br />
<br><br />
We used the following strains, all in the ''E. coli'' strain MG1655. To quantify the leakiness in terms of expression of the Kan<sup>*</sup> gene, we performed real time experiment, where we measured the growth rate (through OD<sub>600</sub> measurement) of each of these strains in different concentrations of kanamycin : <br />
<br />
(1) '''pSB1A1::Kan<sup>R</sup> + pSB1C3::supD''':<br> positive control : Expresses constitutively kanamycine resistance gene.<br />
<br />
(2) '''pSB1A1::Kan<sup>*</sup> + pSB1C3::supD''':<br> construction : Is supD as efficient as the positive control?<br />
<br />
(3) '''pSB1A1::Kan<sup>*</sup> + pSB1C3::RFP''':<br> construction : Is Kan* as unefficient as the negative control?<br />
<br />
(4) '''pSB1A2::P<sub>Lac</sub> + pSB1C3::supD''':<br> negative control : No kanamycin resistance gene.<br />
<br />
In case of leakiness, the Kan* + RFP strain (3) will be able to grow in higher concentration of kanamycin than the negative control (4).<br />
<br><br />
<br><br />
<br />
==Experiments and results==<br />
<br />
<br />
===Qualitative characterization of K914000 (supD) and K914009 (Kan*)===<br />
[[File:GraphCFUqual.png|thumb|border=0|500px|right|'''Figure 3 :''' Number of CFU/µg of plasmid after the different concentration]]<br />
<br />
<br />
After preparing electro-competent M1655 cells with either pSB1C3::supD or pSB1C3::RFP. We transform the plasmid pSB1A1::Kan<sup>*</sup> in both competent cells. After transformation, cells are plated on Cm+Kan.<br />
<br />
We can observe that without any plasmids transformed no cells grow, or when we transform another plasmid with no Kan resistance gene, but with an Amp gene resistance (and plated on Cm+Amp), colonies appear in the strain with RFP, unfortunately the other control (with supD) did not work this time, hence there is no picture of it. But for the quantitative experiment we need that control too (4), and we manage to do it that times. But the pSB1A1::Kan<sup>*</sup> can express the kanamycin resistance phenotype only in the strain containing the supD gene.<br />
<br />
We can conclude here that the supD gene can rescue the phenotype Kan<sup>S</sup> by allowing the correct expression of the kan gene P1003.<br />
<br />
<center><br />
<gallery caption="plates of the double transformation" perrow="5"><br />
File:RFP_pur_Cm_Kan.png|MG1655 + pSB1C3::supD only Selection : Cm+Kan<br />
File:RFP_pur_Cm_Kan.jpg|MG1655 + pSB1C3::RFP only Selection : Cm+Kan<br />
File:SupD%2BKan*_pur_Cm_Kan.jpg|MG1655 + pSB1C3::supD + pSB1A1::Kan<sup>*</sup> Selection : Cm+Kan<br />
File:RFP%2BKan*_pur_Cm_Kan.jpg|MG1655 + pSB1C3::RFP + pSB1A1::Kan<sup>*</sup> Selection : Cm+Kan<br />
File:RFP%2BpLac_200uL_Cm_Amp.jpg|MG1655 + pSB1C3::RFP + pSB1A2::pLac<sup>*</sup> Selection : Cm+Amp<br />
</gallery><br />
</center><br />
<br />
===Quantitative characterization of K914000 (supD) and K914009 (Kan*)===<br />
Here we characterize both biobrick quantitatively. First, we are going to confirm the qualitative result for the part K914000 and then we will determine how a single amino-acid substitution is leaky.<br />
====Experimental setup====<br />
Once the construction is made (Fig. 2), we double transformed the plasmids into MG1655 strains, that does not contain any amber suppressor. We will work with three replicates of each strains, and for each strain we will be in 8 different conditions of antibiotic resistance. The antibiotic used is kanamycin, an aminoglycoside interfering with the translation. The range of concentration goes from 4 times as much as the usual concentration (100 µg/mL) to 8 times less. The 96 wells plate is then incubated in a plate reader that take measurements of OD<sub>600</sub> every 6 minutes. This measure is correlated to the number of cell in the well. <br />
Each well contains 200µL of LB (Lysogeny broth, aka Luria Bertani), chloramphenicol and ampicilin at their usual concentration, the dilution of cells and different amount of kanamycin. An overlay of 50µL of mineral oil is added on the top. The measurement lasted approximately 16 hours and 30 min.<br />
[[File:SCquantitativec.png|thumb|center|800px|'''Figure 4 :''' The over night (O/N) culture is diluted twice, first to normalize all samples, and then to start with low concentration of cells.]]<br />
<br />
====Results====<br />
<br />
First we observed that the qualitative result is reproduce here, the supD gene rescues the kanamycin resistance at any concentration of the antibiotic. It shows also that there is no disadvantage to use the supD amber suppressor compared to the wild type kanamycin gene resistance, since there is no difference of growth rate (Figure 4B and 4C). However the leakiness of the Kan* gene is higher than expected, and thus one mutation is not sufficient for the containment. Indeed, at usual concentration, Kan* gene manages to express an antibiotic resistance, even though lower than Kan<sup>R</sup> or supD (Figure 4A). Here we define the growth rate as the OD<sub>600</sub> observed at a given time (here t=8h20'), in order to overcome the fact that Kan*+RFP does not have a clear exponential phase. The fact that the strain (3) grows faster and can have higher OD<sub>600</sub> may be explain by the fact that supD is deleterious for the strain, by removing some stop codon of other genes for instance. <br />
<br />
[[File:SCquantitativeF1a.png|thumb|center|800px|'''Figure 4A and 4B:''' The bar graph represent the OD<sub>600</sub> at time = 8h20' (black line Figure 4C) for the different strains at a given kanamycin concentration (black line on Figure 4B). On B, we observe the variations of the OD<sub>600</sub> at different kanamycin concentrations, at the same time.]]<br />
<br />
[[File:SCquantitativeF2.png|thumb|center|800px|'''Figure 4C:''' The variations of the OD<sub>600</sub> is observed in function of the time, for different concentrations of kanamycin (400µg/mL, 100µg/mL, 25µg/mL).]]<br />
<br />
<div id="boston"><br />
===Quantitative characterization of K914018 (Kan**)===<br />
Here we perform the exact same experiment than the previous one, except that kanamycin resistant gene is contained with two amber mutations instead of one. It is observed that the positive and negative controls behave correctly, and similarly to the previous experiment, it is also the case for the complementation of Kan** with supD. That means that two mutation are not a problem for the cell. However, we notice that there is no more leakiness in the system at usual concentration, even at low concentration. Actually the double kanamycin resistant gene with two mutation behave exactly like the negative control (Plac + supD) which means that semantic containment is working well.<br />
<br />
[[File:SCquantitative**2.png|thumb|center|800px|'''Figure 5A and 5B:''' The bar graph represent the OD<sub>600</sub> at time = 8h37' (black line Figure 5C) for the different strains at a given kanamycin concentration (black line on Figure 5B). On B, we observe the variations of the OD<sub>600</sub> at different kanamycin concentrations, at the same moment.]]<br />
<br />
[[File:SCquantitative**1.png|thumb|center|801px|'''Figure 5C:''' The variations of the OD<sub>600</sub> is observed in function of the time, for different concentrations of kanamycin (400µg/mL, 100µg/mL, 25µg/mL and 3.125µg/mL).]]<br />
<br />
=== Comparison between one and two mutation ===<br />
Here is a graph that gather both results, to highlight the effect of the second mutation. The results are normalized intra group (experiment with the one-mutated gene, and with the two-mutated gene) in order to have the same scale to compare them. We took the average of the positive (KanR+supD) and the negative (Plac+supD) controls. <br />
[[File:SCquantitative**bilan.png|thumb|center|800px|'''Figure 6:''' Variation of OD<sub>600</sub> in different concentrations of [Kan] (µg/mL) at t= 8,37h]]<br />
<br />
</div><br />
<br />
==Conclusion & Perspectives==<br />
<br />
This work demonstrates that semantic containment can be achieved by changing an amino-acid into an amber codon. We saw that one codon replacement is not enough because of some leakiness, and demonstrated that upon the replacement of two codons, the system isn't leaky at all. It means that for a robust semantic containment system we need 3 codon replacements, because with two replacements we are only one mutation away of a leaky expression. The underlying idea is ''in fine'' to create a library of semantic-contained genes and backbones that would be improved by anybody, by either adding new semantic systems, or increasing the degree of containment by adding mutations on already existing parts. This library is already started with the previously described part and with a plasmid backbone that has only one mutation so far. But we also showed that tRNA amber suppressor might be deleterious for the cell. This trade-off between the leakiness and the toxicity has to be studied in order to optimize the system.<br />
<br />
Further experiments should improve the robustness of this system by adding a new security component that would ensure that as much as three genes would have to be transferred by HGT, to express synthetic gene in natural bacteria, thus cousing our containment system to fail. In our current design, the system fails if the tRNA amber suppressor gene is transferred with another semantic gene into a natural bacteria. The idea is to add a semantic containment for the tRNA. Since it is a tRNA, we cannot replace amino-acids because it is not translated. In order to fix that problem we will construct the following system. The tRNA supD gene will be under a T7 promoter which is orthogonal, meaning that it needs a special RNA polymerase (T7 RNA polymerase) to transcribe the gene (here it would be supD), but we would mutate the T7 RNA polymerase with several amber mutations. It means that we would need to transfer these two genes with the semantically contained gene in order to have something functional in the other organism, which is very unlikely to happen. Further experiments will elucidate the probability of such an event. A scheme of this system is depicted in Figure 5. This system needs to be activated in the lab, by transforming a plasmid with the wild type T7 RNA polymerase gene with another antibiotic gene resistance, say X. Then we would remove antibiotic X and loose the plasmid with wild type T7 polymerase when the positive feedback loop starts.<br />
<br />
[[File:SCT7supD.png|thumb|800px|center|'''Figure 5 : '''Once the mRNA of the T7 RNA polymerase is transcribed (A), it needs the tRNA amber suppressor (B) to let the ribosome translate the mRNA into a functional protein. Then the T7 RNA polymerase will be able to transcribe the supD gene into this tRNA amber suppressor (C). Then it is a positive feedback loop, we need to start this system by adding either tRNA in the medium or T7 RNA pol wild type gene.]]<br />
<br />
==References & Appendix==<br />
===References===<br />
[[#Overview|1]] - Marliere, P. The farther, the safer : a manifesto for securely navigating synthetic species away from the old living world. System and Synthetic Biology 3, 77-84 (2009). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759432/ Paper]<br />
<br />
[[#Overview|2]] - Isaacs, F.J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science (New York, N.Y.) 333, 348-53 (2011). [http://arep.med.harvard.edu/pdf/Isaacs_Sci_11.pdf Paper]<br />
<br />
[[#Overview|3]] - Anderson, J.C., Voigt, C. a & Arkin, A.P. Environmental signal integration by a modular AND gate. Molecular systems biology 3, 133 (2007). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1964800/ Paper]<br />
<br />
[[#Calculation_of_serine_and_tyrosine_weakness|4]] - S Henikoff and J G Henikoff. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A. 1992 November 15; 89(22): 10915–10919. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC50453/pdf/pnas01096-0363.pdf Paper]<br />
<br />
[[#Calculation_of_serine_and_tyrosine_weakness|5]] - Jorja G. Henikoff, Steven Henikoff, Blocks database and its applications, Methods in Enzymology, Academic Press, 266, 88-105 (1996). [http://www.sciencedirect.com/science/article/pii/S007668799666008X Paper]<br />
<br />
===Appendix===<br />
====Genotype of strain used====<br />
<br />
* MG1655 : F- λ- ilvG- rfb-50 rph-1 <br />
<br />
==== Other amino-acids weakness score ====<br />
<br />
* Weakness score with a BLOSUM 100, mutation rate = 10<sup>-9</sup>, initial Codon : TAG<br />
<br />
[[#Weakness_calculation|main result part]]<br />
<br />
('A', [-4.000000002555556e-09])<br />
<br />
('C', [-6.777777782148149e-09])<br />
<br />
('D', [-5.2222222257407416e-09])<br />
<br />
('E', [-3.222222225407407e-09])<br />
<br />
('F', [-3.0000000040740746e-09])<br />
<br />
('G', [-6.555555560444445e-09])<br />
<br />
('H', [-2.888888891555555e-09])<br />
<br />
('I', [-5.111111115777778e-09])<br />
<br />
('K', [-2.6666666694074067e-09])<br />
<br />
('L', [-4.333333337555555e-09])<br />
<br />
('M', [-4.000000003185186e-09])<br />
<br />
('N', [-4.3333333354074076e-09])<br />
<br />
('P', [-5.888888893518519e-09])<br />
<br />
('Q', [-1.6666666689629633e-09])<br />
<br />
('R', [-3.888888891888888e-09])<br />
<br />
('S', [-3.333333334925926e-09])<br />
<br />
('T', [-4.22222222462963e-09])<br />
<br />
('V', [-4.888888893111112e-09])<br />
<br />
('W', [-2.4444444508518513e-09])<br />
<br />
('Y', [-1.0000000044444444e-09])<br />
<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Semantic_containmentTeam:Paris Bettencourt/Semantic containment2012-10-27T00:31:01Z<p>Aleksandra: /* Conclusion & Perspectives */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<div id="grouptitle">Semantic containment</div><br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Aims'''<br />
* Creating a semantic containment system to prevent gene expression in natural organisms<br />
* Characterize the system<br />
* Use this system in all genes of the system, the critical genes first (e.g. [https://2012.igem.org/Team:Paris_Bettencourt/Suicide colicin])<br />
<br />
'''System'''<br />
*An amber codon (stop codon) embedded in protein genes to prevent their expression and an amber suppressor system in our genetically engineered bacteria<br />
<br />
'''Achievements :'''<br />
* Construction and characterization of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914000 K914000] : P<sub>Lac</sub>-supD-T : tRNA amber suppressor<br />
** [http://partsregistry.org/Part:BBa_K914009 K914009] : P1003* Ser133->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
<br />
Both part were well [[#Results|characterized]] and works well. For the second parts, we show that as expected, one mutation is quite leaky, although it works qualitatively, but one mutation is not enough if we want to release such parts in nature. Other reasons emphasize this observation, notably the weakness of being at one mutation to recover the protein functionality.<br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety Semantic containment]. The aim of this category is to let people improving each part by adding for instance other amber mutations to existing part to increase the containment.<br />
<div id="boston"><br />
'''Achievements : '''<br />
* Construction and characterization of 1 biobrick :<br />
** [http://partsregistry.org/Part:BBa_K914018 K914018] : P1003** Ser133 & Ser203 ->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
* Construction of 1 plasmid backbone : <br />
** [http://partsregistry.org/Part:BBa_K914012 K914012] : pSB1A2 with one Amber Codon : Ampicillin gene resistance with 1 amber mutation<br />
</div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
==Overview==<br />
We want to prevent our genetic construct from conferring an advantage to other organisms or alter them phenotypically. Minimizing horizontal gene transfer (HGT), either by conjugation, transduction, or transformation is thus our main concern. As these processes involve two parties, the genetically modified bacteria and some wild type population partners, and as we will not modify wild type populations, we cannot assume that HGT is fully avoidable. Semantic containment<sup>[</sup><sup>[[#References|1]]</sup><sup>]</sup> means that our bacteria won't be able to "speak" with other organisms, since they don't speak the same "language", the language being DNA. Our system will read the stop codon TAG as the amino-acid serine. It means that in our bacteria the stop codon will be translated into a serine, whereas in wild type bacteria this protein will be truncated and will not confer an advantage to these cells. The 'TAG' codon has been chosen because of its low frequency in ''E. coli'' genome (314 occurrences), and also because for further applications, Church Lab tries to remove all amber codon of an ''E. coli'' strain<sup>[</sup><sup>[[#References|2]]</sup><sup>]</sup>. Although it has been demonstrate that the over-expression of a tRNA amber suppressor sole does not affect its growth rate nor the morphology of ''E. coli''<sup>[</sup><sup>[[#References|3]]</sup><sup>]</sup>.<br />
[[File:ConceptSC.png|thumb|center|800px|'''Figure 1 :''' Semantic containment principle. The information carried by the DNA cannot be read by other organism]]<br />
<br />
==Objectives==<br />
We want to create a new way to contain semantically genes, by replacing an amino-acid codon by a stop codon (amber codon, 'TAG'), and in our synthetic cell this stop codon will be read as the amino-acid. Here we want to show that this system works as expected. First we had to choose between two tRNA amber suppressor, either serine, or tyrosine that are available in the part registry. For that we calculate the abilities for the amber codon to reverse to a serine or tyrosine or related amino-acid that could conserve the function. Secondly we create a biobrick with a tRNA Amber suppressor ([http://partsregistry.org/Part:BBa_K914000 BBa_K914000]), in order to have a reliable biobrick, with characterization of it. Thirdly, to test the latter biobrick, we built a biobrick which is a kanamycin resistance gene (P1003) with one amber mutation added instead of the serine 133 ([http://partsregistry.org/Part:BBa_K914009 BBa_K914009]).<br />
<br />
K914000 is the construction P<sub>Lac</sub>-supD-T, and is named supD in the rest of the page. K914009 is the P1003 gene (kanamycin resistance gene) with the serine 133 which is replaced by a amber codon 'TAG'. Its name is Kan<sup>*</sup>.<br />
<br />
With more time we will try to increase the robustness of this system, which is null when the tRNA amber suppressor is transferred too. We will try to create a new library of plasmid backbones in the part registry, where all backbones have at least two amber mutations. The idea is that all the community will be able to improve this library, either by adding new contained backbones, or by adding amber mutations on the same backbone, or add semantic containment to any other gene.<br />
<br />
==Design==<br />
===How to choose between serine and tyrosine ? ===<br />
At one mutation of the 'TAG' codon we can have one serine or two tyrosine (among others). Even if the serine seems more interesting by this simple observation, we still want to know which of these two amino-acids is the less robust to mutation from a 'TAG' codon (amber codon) including similarity property of amino-acids. We calculated a score of weakness. The weakness of an amino-acid is defined here by its abilities to not revert to the same amino-acid or any other similar, from the amber codon.<br />
The score is calculated using the following formula :<br />
<center><br />
<html><br />
<img src="http://www.openwetware.org/images/math/6/7/3/673fca1559040fa2561513142404b963.png"><br />
</html><br />
</center><br />
<br />
Where i is one of the nine amino-acids accessible after 1 mutation. Subst(AA,AAi) calculate the similarity score, using a BLOSUM100 matrix, between serine or tyrosine (AA) and one of the nine amino-acids around (AAi). The lowest score is the weakest. The BLOSUM100 (BLOck SUbstitution Matrix) is constructed using local alignment of sequenced less than 100% identical<sup>[</sup><sup>[[#References|4,5]]</sup><sup>]</sup>, and is consequently adapted to appreciate the effect of a single mutation.<br />
<br />
===Weakness calculation===<br />
<br />
We wrote a Python script that can calculate the score of weakness, with all amino-acids (not only serine or tyrosine), and will sort a list of score. Data shown in [[#Other_amino-acids_weakness_score|appendix]] for other amino-acids.<br />
<br />
The scores are, with a mutation rate of 10<sup>-9</sup> for the serine and tyrosine:<br />
<br />
*BLOSUM100 :<br />
** Serine : Score<sup>S</sup><sub>W</sub> = -3.33e-09<br />
** Tyrosine : Score<sup>Y</sup><sub>W</sub> = -1.00e-09<br />
<br />
The amber codon is less likely to revert into a serine or similar.<br />
<br />
Also, we favored serine replacement over tyrosine, because the frequency of serine in the ''E. coli'' genome is superior to the tyrosine one. It turns out that S has 57,88 codons over 1000 codons when Y has 28,59 codons over 1000 codons ([http://openwetware.org/wiki/Escherichia_coli/Codon_usage Codon usage]). Therefor, it might be more convenient to replace a serine than a tyrosine, because proteins would more likely to contain many serines than tyrosines.<br />
<br />
===Can the tRNA rescues the Kan<sup>R</sup> phenotype ? (Qualitative experiment)===<br />
[[File:SCsupD.gif|thumb|center|800px|'''Figure 3 :''' Our hypothesis : supD can rescue the Kan<sup>R</sup> phenotype. '''A''' production of the mRNA from the Kan* gene. '''B''' The mRNA is translated only if there is a tRNA amber suppressor (supD)]]<br />
<br />
To do so, we will transform a plasmid with a the Kan<sup>*</sup> gene into a MG1655 strain that contain either pSB1C3::supD, or pSB1C3::RFP. We plate them on Chloramphenicol and Kanamycin. The Kan<sup>*</sup> is supposed to be non functional without an amber suppressor.<br />
<br />
===Is it working well? Is the amber mutation leaky? (Quantitative experiment)===<br />
[[File:Design1SC.png|thumb|right|600px| '''Figure 2 :''' Schema of the four strains used for the quantitative experiments]]<br />
<br><br />
<br><br />
We used the following strains, all in the ''E. coli'' strain MG1655. To quantify the leakiness in terms of expression of the Kan<sup>*</sup> gene, we performed real time experiment, where we measured the growth rate (through OD<sub>600</sub> measurement) of each of these strains in different concentrations of kanamycin : <br />
<br />
(1) '''pSB1A1::Kan<sup>R</sup> + pSB1C3::supD''':<br> positive control : Expresses constitutively kanamycine resistance gene.<br />
<br />
(2) '''pSB1A1::Kan<sup>*</sup> + pSB1C3::supD''':<br> construction : Is supD as efficient as the positive control?<br />
<br />
(3) '''pSB1A1::Kan<sup>*</sup> + pSB1C3::RFP''':<br> construction : Is Kan* as unefficient as the negative control?<br />
<br />
(4) '''pSB1A2::P<sub>Lac</sub> + pSB1C3::supD''':<br> negative control : No kanamycin resistance gene.<br />
<br />
In case of leakiness, the Kan* + RFP strain (3) will be able to grow in higher concentration of kanamycin than the negative control (4).<br />
<br><br />
<br><br />
<br />
==Experiments and results==<br />
<br />
<br />
===Qualitative characterization of K914000 (supD) and K914009 (Kan*)===<br />
[[File:GraphCFUqual.png|thumb|border=0|500px|right|'''Figure 3 :''' Number of CFU/µg of plasmid after the different concentration]]<br />
<br />
<br />
After preparing electro-competent M1655 cells with either pSB1C3::supD or pSB1C3::RFP. We transform the plasmid pSB1A1::Kan<sup>*</sup> in both competent cells. After transformation, cells are plated on Cm+Kan.<br />
<br />
We can observe that without any plasmids transformed no cells grow, or when we transform another plasmid with no Kan resistance gene, but with an Amp gene resistance (and plated on Cm+Amp), colonies appear in the strain with RFP, unfortunately the other control (with supD) did not work this time, hence there is no picture of it. But for the quantitative experiment we need that control too (4), and we manage to do it that times. But the pSB1A1::Kan<sup>*</sup> can express the kanamycin resistance phenotype only in the strain containing the supD gene.<br />
<br />
We can conclude here that the supD gene can rescue the phenotype Kan<sup>S</sup> by allowing the correct expression of the kan gene P1003.<br />
<br />
<center><br />
<gallery caption="plates of the double transformation" perrow="5"><br />
File:RFP_pur_Cm_Kan.png|MG1655 + pSB1C3::supD only Selection : Cm+Kan<br />
File:RFP_pur_Cm_Kan.jpg|MG1655 + pSB1C3::RFP only Selection : Cm+Kan<br />
File:SupD%2BKan*_pur_Cm_Kan.jpg|MG1655 + pSB1C3::supD + pSB1A1::Kan<sup>*</sup> Selection : Cm+Kan<br />
File:RFP%2BKan*_pur_Cm_Kan.jpg|MG1655 + pSB1C3::RFP + pSB1A1::Kan<sup>*</sup> Selection : Cm+Kan<br />
File:RFP%2BpLac_200uL_Cm_Amp.jpg|MG1655 + pSB1C3::RFP + pSB1A2::pLac<sup>*</sup> Selection : Cm+Amp<br />
</gallery><br />
</center><br />
<br />
===Quantitative characterization of K914000 (supD) and K914009 (Kan*)===<br />
Here we characterize both biobrick quantitatively. First, we are going to confirm the qualitative result for the part K914000 and then we will determine how a single amino-acid substitution is leaky.<br />
====Experimental setup====<br />
Once the construction is made (Fig. 2), we double transformed the plasmids into MG1655 strains, that does not contain any amber suppressor. We will work with three replicates of each strains, and for each strain we will be in 8 different conditions of antibiotic resistance. The antibiotic used is kanamycin, an aminoglycoside interfering with the translation. The range of concentration goes from 4 times as much as the usual concentration (100 µg/mL) to 8 times less. The 96 wells plate is then incubated in a plate reader that take measurements of OD<sub>600</sub> every 6 minutes. This measure is correlated to the number of cell in the well. <br />
Each well contains 200µL of LB (Lysogeny broth, aka Luria Bertani), chloramphenicol and ampicilin at their usual concentration, the dilution of cells and different amount of kanamycin. An overlay of 50µL of mineral oil is added on the top. The measurement lasted approximately 16 hours and 30 min.<br />
[[File:SCquantitativec.png|thumb|center|800px|'''Figure 4 :''' The over night (O/N) culture is diluted twice, first to normalize all samples, and then to start with low concentration of cells.]]<br />
<br />
====Results====<br />
<br />
First we observed that the qualitative result is reproduce here, the supD gene rescues the kanamycin resistance at any concentration of the antibiotic. It shows also that there is no disadvantage to use the supD amber suppressor compared to the wild type kanamycin gene resistance, since there is no difference of growth rate (Figure 4B and 4C). However the leakiness of the Kan* gene is higher than expected, and thus one mutation is not sufficient for the containment. Indeed, at usual concentration, Kan* gene manages to express an antibiotic resistance, even though lower than Kan<sup>R</sup> or supD (Figure 4A). Here we define the growth rate as the OD<sub>600</sub> observed at a given time (here t=8h20'), in order to overcome the fact that Kan*+RFP does not have a clear exponential phase. The fact that the strain (3) grows faster and can have higher OD<sub>600</sub> may be explain by the fact that supD is deleterious for the strain, by removing some stop codon of other genes for instance. <br />
<br />
[[File:SCquantitativeF1a.png|thumb|center|800px|'''Figure 4A and 4B:''' The bar graph represent the OD<sub>600</sub> at time = 8h20' (black line Figure 4C) for the different strains at a given kanamycin concentration (black line on Figure 4B). On B, we observe the variations of the OD<sub>600</sub> at different kanamycin concentrations, at the same time.]]<br />
<br />
[[File:SCquantitativeF2.png|thumb|center|800px|'''Figure 4C:''' The variations of the OD<sub>600</sub> is observed in function of the time, for different concentrations of kanamycin (400µg/mL, 100µg/mL, 25µg/mL).]]<br />
<br />
<div id="boston"><br />
===Quantitative characterization of K914018 (Kan**)===<br />
Here we perform the exact same experiment than the previous one, except that kanamycin resistant gene is contained with two amber mutations instead of one. It is observed that the positive and negative controls behave correctly, and similarly to the previous experiment, it is also the case for the complementation of Kan** with supD. That means that two mutation are not a problem for the cell. However, we notice that there is no more leakiness in the system at usual concentration, even at low concentration. Actually the double kanamycin resistant gene with two mutation behave exactly like the negative control (Plac + supD) which means that semantic containment is working well.<br />
<br />
[[File:SCquantitative**2.png|thumb|center|800px|'''Figure 5A and 5B:''' The bar graph represent the OD<sub>600</sub> at time = 8h37' (black line Figure 5C) for the different strains at a given kanamycin concentration (black line on Figure 5B). On B, we observe the variations of the OD<sub>600</sub> at different kanamycin concentrations, at the same moment.]]<br />
<br />
[[File:SCquantitative**1.png|thumb|center|801px|'''Figure 5C:''' The variations of the OD<sub>600</sub> is observed in function of the time, for different concentrations of kanamycin (400µg/mL, 100µg/mL, 25µg/mL and 3.125µg/mL).]]<br />
<br />
=== Comparison between one and two mutation ===<br />
Here is a graph that gather both results, to highlight the effect of the second mutation. The results are normalized intra group (experiment with the one-mutated gene, and with the two-mutated gene) in order to have the same scale to compare them. We took the average of the positive (KanR+supD) and the negative (Plac+supD) controls. <br />
[[File:SCquantitative**bilan.png|thumb|center|800px|'''Figure 6:''' Variation of OD<sub>600</sub> in different concentrations of [Kan] (µg/mL) at t= 8,37h]]<br />
<br />
</div><br />
<br />
==Conclusion & Perspectives==<br />
<br />
This work demonstrates that semantic containment can be achieved by changing an amino-acid into an amber codon. We saw that one mutation is not enough because of some leakiness, and demonstrated that two mutations is not leaky at all. It means that for a robust semantic containment system we need 3 codon replacements, because with two replacements we are only one mutation away of a leaky expression. The underlying idea is ''in fine'' to create a library of semantic-contained genes and backbones that would be improved by anybody, by either adding new semantic systems, or increasing the degree of containment by adding mutations on already existing parts. This library is already started with the previously described part and with a plasmid backbone that has only one mutation so far. But we also showed that tRNA amber suppressor might be deleterious for the cell. This trade-off between the leakiness and the toxicity has to be studied in order to optimize the system.<br />
<br />
Further experiments should improve the robustness of this system by adding a new security component that would ensure that as much as three genes would have to be transferred by HGT, to express synthetic gene in natural bacteria, thus cousing our containment system to fail. In our current design, the system fails if the tRNA amber suppressor gene is transferred with another semantic gene into a natural bacteria. The idea is to add a semantic containment for the tRNA. Since it is a tRNA, we cannot replace amino-acids because it is not translated. In order to fix that problem we will construct the following system. The tRNA supD gene will be under a T7 promoter which is orthogonal, meaning that it needs a special RNA polymerase (T7 RNA polymerase) to transcribe the gene (here it would be supD), but we would mutate the T7 RNA polymerase with several amber mutations. It means that we would need to transfer these two genes with the semantically contained gene in order to have something functional in the other organism, which is very unlikely to happen. Further experiments will elucidate the probability of such an event. A scheme of this system is depicted in Figure 5. This system needs to be activated in the lab, by transforming a plasmid with the wild type T7 RNA polymerase gene with another antibiotic gene resistance, say X. Then we would remove antibiotic X and loose the plasmid with wild type T7 polymerase when the positive feedback loop starts.<br />
<br />
[[File:SCT7supD.png|thumb|800px|center|'''Figure 5 : '''Once the mRNA of the T7 RNA polymerase is transcribed (A), it needs the tRNA amber suppressor (B) to let the ribosome translate the mRNA into a functional protein. Then the T7 RNA polymerase will be able to transcribe the supD gene into this tRNA amber suppressor (C). Then it is a positive feedback loop, we need to start this system by adding either tRNA in the medium or T7 RNA pol wild type gene.]]<br />
<br />
==References & Appendix==<br />
===References===<br />
[[#Overview|1]] - Marliere, P. The farther, the safer : a manifesto for securely navigating synthetic species away from the old living world. System and Synthetic Biology 3, 77-84 (2009). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759432/ Paper]<br />
<br />
[[#Overview|2]] - Isaacs, F.J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science (New York, N.Y.) 333, 348-53 (2011). [http://arep.med.harvard.edu/pdf/Isaacs_Sci_11.pdf Paper]<br />
<br />
[[#Overview|3]] - Anderson, J.C., Voigt, C. a & Arkin, A.P. Environmental signal integration by a modular AND gate. Molecular systems biology 3, 133 (2007). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1964800/ Paper]<br />
<br />
[[#Calculation_of_serine_and_tyrosine_weakness|4]] - S Henikoff and J G Henikoff. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A. 1992 November 15; 89(22): 10915–10919. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC50453/pdf/pnas01096-0363.pdf Paper]<br />
<br />
[[#Calculation_of_serine_and_tyrosine_weakness|5]] - Jorja G. Henikoff, Steven Henikoff, Blocks database and its applications, Methods in Enzymology, Academic Press, 266, 88-105 (1996). [http://www.sciencedirect.com/science/article/pii/S007668799666008X Paper]<br />
<br />
===Appendix===<br />
====Genotype of strain used====<br />
<br />
* MG1655 : F- λ- ilvG- rfb-50 rph-1 <br />
<br />
==== Other amino-acids weakness score ====<br />
<br />
* Weakness score with a BLOSUM 100, mutation rate = 10<sup>-9</sup>, initial Codon : TAG<br />
<br />
[[#Weakness_calculation|main result part]]<br />
<br />
('A', [-4.000000002555556e-09])<br />
<br />
('C', [-6.777777782148149e-09])<br />
<br />
('D', [-5.2222222257407416e-09])<br />
<br />
('E', [-3.222222225407407e-09])<br />
<br />
('F', [-3.0000000040740746e-09])<br />
<br />
('G', [-6.555555560444445e-09])<br />
<br />
('H', [-2.888888891555555e-09])<br />
<br />
('I', [-5.111111115777778e-09])<br />
<br />
('K', [-2.6666666694074067e-09])<br />
<br />
('L', [-4.333333337555555e-09])<br />
<br />
('M', [-4.000000003185186e-09])<br />
<br />
('N', [-4.3333333354074076e-09])<br />
<br />
('P', [-5.888888893518519e-09])<br />
<br />
('Q', [-1.6666666689629633e-09])<br />
<br />
('R', [-3.888888891888888e-09])<br />
<br />
('S', [-3.333333334925926e-09])<br />
<br />
('T', [-4.22222222462963e-09])<br />
<br />
('V', [-4.888888893111112e-09])<br />
<br />
('W', [-2.4444444508518513e-09])<br />
<br />
('Y', [-1.0000000044444444e-09])<br />
<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/DelayTeam:Paris Bettencourt/Delay2012-10-27T00:10:06Z<p>Aleksandra: /* Characterization */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
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<br />
<div id="grouptitle">Delay system(s)</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Aim :'''<br />
A programmed delay will allow the cell to perform its intended function before our DNA-degrading suicide machinery is expressed.<br />
<br />
'''Experimental system:'''<br />
We used two different approaches to create this delay. The [https://2012.igem.org/Team:Paris_Bettencourt/Delay#Simple_delay_system first one] is based on the gradual dilution of a regulatory transcription factor. The [https://2012.igem.org/Team:Paris_Bettencourt/Delay#sRNA_delay_system second one] makes use of a stationary-phase specific promoter. Both systems eventually result in the expression of the restriction enzyme I-SceI. In the final design, I-SceI cleaves the antitoxin gene, ultimately dooming the cell. Each step in this causal sequence contributes to the overall delay in the system.<br />
<br />
<br />
'''Achievements :'''<br />
* Construction and characterization of the dilution delay system<br />
** [http://partsregistry.org/Part:BBa_K914004 K914004] : P<sub>BAD</sub>-AraC-RBS-LacI ; <br />
** [http://partsregistry.org/Part:BBa_K914014 K914014] : P<sub>BAD</sub>-AraC-RBS-LacI-P<sub>lac</sub>-RBS-GFP-double terminator ; [https://2012.igem.org/Team:Paris_Bettencourt/Delay#Characterization characterization]<br />
** [http://partsregistry.org/Part:BBa_K914015 K914015] : P<sub>BAD</sub>-AraC-RBS-LacI-P<sub>lac</sub><br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Delay#Refined_characterization_of_the_Yokobayashi_et_al._sRNA_repression_plasmidic_device Characterization] of the sRNA repression system of Yokobayashi ''et al.'' <br />
* Cloning of the yiaGp stationary phase promoter<br />
<div id="boston"><br />
* Partially biobricked sRNA system : <br />
**[http://partsregistry.org/Part:BBa_K914017 K914017] stationary phase promoter Yiagp<br />
**[http://partsregistry.org/Part:BBa_K914016 K914016] coding sequence of Colicin E2<br />
</div><br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
==Overview==<br />
The delay system serves to suppress the function of the suicide device and preserve the integrity of the host genome while the organism does its intended job in the environment. We experimented with two designs for producing a programmed delay before the expression of the DNA-degrading suicide machinery.<br />
<br />
The dilution delay system relies on the lab-specific expression of a transcriptional inhibitor. This inhibitor is induced by a specific compound found in the laboratory but not in the environment. When the cell enters the environment the inhibitor is gradually degraded or diluted by cell growth. Eventually, the repressor concentration falls below a critical threshold, releasing the suicide machinery.<br />
<br />
The sRNA delay system makes use of a stationary-phase specific promoter. Under laboratory conditions, the suicide machinery is repressed by an inducible sRNA. In the environment, the suicide machinery is expressed as soon as the cells reach stationary phase.<br />
<br />
[[File:Paris_Bettencourt_Delay_overview.png|frameless|center|600px]]<br />
<br />
==Dilution delay system==<br />
===Objectives===<br />
Our cells need time to work in the environment before we degrade their genomes. A programmed delay circuit faces the following challenges:<br />
<br />
*The delay must be programmable and long enough for our cells to perform a useful task.<br />
*Once begun, the delay countdown should lead inevitably to death, and should not be reversible.<br />
*Prior to induction, our suicide machinery must be very tightly repressed.<br />
<br />
===Design===<br />
We chose to base our delay on a simple transcriptional network. The arabinose-activated promoter, pBAD, drives the production of the LacI gene. LacI represses a restriction enzyme at the pLac promoter. In the absence or arabinose, the restriction enzyme is eventually expressed, triggering irreversible cell death.<br />
<br />
We chose the pLac promoter because of its reputation for tight repression by the lacI protein. Our final design eventually expresses a restriction enzyme, because the DNA hydrolysis reaction is effectively irreversible. But for the purposes of characterization, we substituted GFP for the restriction enzyme at the end of our transcriptional cascade. <br />
<br />
The system we created to characterize our delay is shown below:<br />
<br />
[[File:ParisBet Simple Delay design.jpg|frameless|center|600px]]<br />
<br />
===Characterization===<br />
[[File:Paris_Bettencourt_2012_Simple_delay.png|frameless|center|700px]]<br />
We characterized the behavior of our transcriptional circuit by first growing cells in the presence of 1% arabinose induction. In this state, LacI expression is highly induced. The cells were then washed, diluted, and characterized in the presence of arabinose, IPTG, or no induction.<br />
<br />
The phase between the 'uninduced' and 'IPTG' curves is the delay from dilution of LacI. After removal of arabinose, LacI remains within the cells but soon becomes inefficient at inhibiting GFP expression as the cells divide. The delay is characterized by also adding IPTG to the system, which activates GFP expression despite presence of LacI.<br />
Here, the delay is about 1 hour.<br />
<br />
==sRNA delay system==<br />
===Objectives===<br />
<br />
As an alternative delayed trigger for our suicide machinery we sought to make use of the natural delay experienced by cells as they approach stationary phase. The design requirements for this system are similar to those described above.<br />
<br />
*The delay must be programmable and sufficiently long.<br />
*Death must follow inevitably one the countdown begins.<br />
*The suicide machinery is highly toxic, and must not be expressed until needed.<br />
<br />
===Design===<br />
To achieve tight repression, our system is augmented with an sRNA. The concept of our design is described below. Similar systems have been shown to respond in a treshold-linear way [2] <br />
[[File:sRNAparisbett.png|frameless|center|600px]]<br />
We adapted the construct of Yokobayashi ''et al.'' [1] described below.<br />
[[File:odparisbett.png|frameless|center|600px]]<br />
The final design of our construct is shown below.<br />
[[File:finaldesignparisbett.png|frameless|center|600px]]<br />
<br />
The transcription of the toxin is controlled twice: first by the stationary phase promoter of yiaG. It is recognized for transcription only by the sigma-S subunit of RNA polymerase, and not the exponential phase sigma-70 subunit. [3], [4].<br />
<br />
We have shown that the yiaGp promoter is indeed activated during the stationary phase in our construct, even though the level of trancription is rather low (see experiments)<br />
<br />
In order to achieve a complete toxin lockdown, we added a second post-transcriptional repression mechanism. We used and modified the constructs of Yokobayashi ''et al.'' to allow a repression of the translation using sRNA [1]. Our specific sRNA binds the leader sequence of its target mRNA and not its coding sequence, and allows a 20 fold repression of protein expression. The transcription of the sRNA is under the control of the pBAD promoter.<br />
<br />
We expect the final sRNA delay system will allow the DNA-degradation machinery to be expressed only by cells in stationary phase and lacking arabinose.<br />
<br />
===Cloning of the YiaGp promoter===<br />
<br />
YiaGp is a late stationary phase promoter. It has been shown to be activated by 10 to 30 fold during to stationary phase, and is among the strongest promoters during this phase. Furthermore, its action has been reported to last for a longer time than most stationary phase promoters. [4]<br />
We managed to replace the P33 promoter with the YiaGp promoter in the pKP33-aOmpF-GFPuv plasmid between the XhoI and EcoRI sites. The sequencing was consistent with the expected sequence. However, the way the original plasmids were designed made them difficult to biobrick (presence of restriction sites in the sequences). '''We will try to biobrick the entire design in order to both facilitate the cloning and share our construct with the iGEM community.'''<br />
[[File:GFP-YiaGp.png|frameless|center|400px]]<br />
We still need to characterize this promoter and compare its activity in exponential and stationary phase.<br />
<br />
===Characterization of the Yokobayashi ''et al.'' sRNA repression device===<br />
<br />
We double transformed TOP10 cells with the GFP and pBAD-sRNA plasmids (see [1] for details, and click [https://static.igem.org/mediawiki/2012/c/c4/Odparisbett.png here] for a network schematic). We chose the this sRNA because it shows a strong repression GFP expression, and binds specifically 5' leader sequence. It does not bind the GFP coding sequence, suggesting is could easily adapted for use with other genes.<br />
<br />
The cells were grown in LB until stationary phase, then diluted 100-fold in LB. After adding defined arabinose concentrations to the medium, we monitored the GFP fluorescence with a TECAN 96-well plate reader. The mean normalized fluorescence after 10 hours is reported below (4 replicates per concentration).<br />
<br />
[[File:Pb tecanara.png|thumb|center|600px|Normalized Fluorescence of GFP with different concentrations of arabinose]]<br />
<br />
= References =<br />
1. Sharma, V., Yamamura, A., & Yokobayashi, Y. (2012). Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli, 6-13.<br />
[http://pubs.acs.org/doi/abs/10.1021/sb200001q| Paper]<br />
<br />
2. Levine, E., Zhang, Z., Kuhlman, T., & Hwa, T. (2007). Quantitative characteristics of gene regulation by small RNA. PLoS biology, 5(9), e229. doi:10.1371/journal.pbio.0050229<br />
[http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0050229| Paper]<br />
<br />
3. Sharma, U. K., & Chatterji, D. (2010). Transcriptional switching in Escherichia coli during stress and starvation by modulation of sigma activity. FEMS microbiology reviews, 34(5), 646-57. doi:10.1111/j.1574-6976.2010.00223.x[http://jb.asm.org/content/186/21/7112.long| Paper]<br />
<br />
4. Shimada, T., Makinoshima, H., Ogawa, Y., Miki, T., Maeda, M., & Ishihama, A. (2004). Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector, 186(21), 7112-7122. doi:10.1128/JB.186.21.7112 [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2010.00223.x/abstract;jsessionid=8C98B1B383242B9DFF45429802F48428.d02t04| Paper]<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/ModelingTeam:Paris Bettencourt/Modeling2012-10-26T23:12:55Z<p>Aleksandra: /* Check controls */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="boston"><br />
<br><br />
<div id="grouptitle">Safety Assessment</div><br />
<br />
==Overview==<br />
Safety is an important issue in synthetic biology, especially on environmental related projects. We already tried to answer the question, “how safe is safe enough?” by involving experts, publics and scientists, and also building biosafety devices. However, to really answer the question, actually we need first to ask ourselves a more basic question, “how do we measure safety?”. As we see synthetic biology as an engineering approach to biology, we could think about adaptation of safety engineering, a well studied engineering subset, that has been widely use to minimize risks on many fields of engineering, such as mechanical engineering, aircrafts, and manufactures. However, the risks they face are surely different from the risks of synthetic biology. <br />
<br />
According to Dana et al<sup>[[#References|1]]</sup> there are four areas of risk research in environmental application of synthetic biology:<br />
*Differences in the physiology of natural and synthetic organisms will affect how they interact with the surrounding environment,<br />
*Escaped microorganisms have the potential to survive in receiving environments and to compete successfully with non-modified counterparts,<br />
*Synthetic organisms might evolve and adapt quickly, perhaps filling new ecological niches, and<br />
*Gene transfer.<br />
<br />
Knowing that there are different areas of risk that we need to take into account, we need to design our safety containment with different parts to deal with each of them. Identifying the relationship between one part and the others will help us to see the reliability of the overall system. Here we propose an approach to assess safety for environmental release of genetically engineered bacteria (GE bacteria).<br />
<br />
===Objectives===<br />
*Adapting existing safety assessment tools to synthetic biology.<br />
*Proposing methods to assess safety in synthetic biology.<br />
<br />
==Methods and Results==<br />
We propose a method to assess hazards and risk<sup>[[#References|2]]</sup> in releasing genetically engineered bacteria to the environment. As a case study, we want to release GE bacteria which will perform some function in the environment and we want to apply 3 sets of bWARE safety containment (alginate beads, bWARE killswitch, semantic containment) to control the hazards and risks. Here we focus more on the reliability of the safety containment system rather than the functional part, although the similar assessment can also be applied to see the functional part reliability.<br />
<br />
===Hazards Identification===<br />
Identifying hazards means that we need to find and understand the possible harm that may happen in the application of our system. Generally there are two potential hazard in environmental application of synthetic biology<sup>[[#References|1]]</sup> which are the success escape of the GE bacteria followed by a success competition with the natural strain and the horizontal gene transfer.<br />
<br />
===Risk Assessment===<br />
Risk assessment will give an idea what kind of risk we face in releasing the GEO in the environment and help to design the safety containment. Here we adapted a risk management method in workplaces complying with health and safety law <sup>[[#References|3]]</sup>.<br />
<br />
To-do-list to assess risk in 5 steps:<br />
#Identify the hazards<br />
#Decide who might be harmed and how<br />
#Evaluate the risks and decide on precaution<br />
#Record your findings and implement them<br />
#Review your assessment and update if necessary<br />
<br />
<br />
Worksheet example:<br />
{| style="border-spacing:0;"<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''What are the hazards?'''<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''Who might be harmed and how?'''<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''What are we already doing?'''<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''What further action is necessary?'''<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| GE bacteria outcompete natural strains<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| GE bacteria escaping the containment may outcompete natural strains if they have better fitness, creating natural imbalance <br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| Using harmless strain or strain with low fitness compared to natural strain (standard E coli for lab)<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| Designing a safety containment to prevent the cells reproducing themselves at some point and/or separating them from natural strains<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| Horizontal gene transfer (HGT)<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| Other strain/species may uptake engineered gene, and if the gene gives advantage in the fitness, it may outcompete other strain and creating natural imbalance<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| -<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| Designing a safety containment to degrade DNA and/or separate the DNA from the environment and/or prevent natural strain having advantages from the DNA<br />
<br />
|}<br />
<br />
<br><br />
===Control hazards and risks===<br />
<br />
In this step we decided what control elements we want to put to our system to reduce the risks. We should choose the best suitable control elements depending on the system function and application. As we already know that we want to apply bWARE safety containment, here we describe the role of each safety parts in the risks reduction. <br />
#GE bacteria outcompete natural strains<br />
#*In order to prevent the success escape of GE bacteria and outcompete the natural strains, we will stop their reproduction by putting a self killing mechanism to kill the cells after they perform the function. <br />
#*In case of the failure of the killing mechanism, we will put the cells inside a physical containment so there will be no physical interaction between them and the surrounding cells.<br />
#HGT<br />
#*We will use DNAse to degrade DNA after the cells perform the function so they won’t leave any genetic material behind.<br />
#*In case of the failure on inefficiency of the DNAse, the physical containment will still protect the DNA from the environment.<br />
#*In case of the leakiness of the physical containment, the DNA has special encryption system with the semantic containment so the receiver cell will not be able to read it.<br />
<br />
===Check controls=== <br />
<br />
To check and predict the reliability of our system, we used fault tree analysis to assess multiple safety containment in one biodevice. This top-down approach allows us to see the relationship between each containment element and predict the overall failure probability. With this method we can see what basic events are the key elements in our system.<br />
<br />
[[File:ParisB_FTA.png|800px|center]]<br />
<br />
AND gate represents independent events<br />
<br />
P(A and B) = P(A)P(B)<br />
<br />
OR gate corresponds to set of union<br />
<br />
P(A or B) = P(A) + P(B)<br />
<br />
so the total failure probability of the system is<br />
<br />
Ptotal = P1.P2.P3.P4.P5 + P1.P6.(P5+P7+P8+P9)<br />
<br />
List of the basic events<br />
<br />
{| style="border-spacing:0;"<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''Notation'''<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''Failure component'''<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''Failure mode'''<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''Consequence'''<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| '''Method for determining the probability'''<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P1<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| physical containment<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| leakage<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| DNA/cell escape<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| experiment<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P2<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| DNA uptake<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| DNA transferred into natural strain<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| HGT<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| <nowiki>transconjugant to donor ratio in HGT is typically <10</nowiki><sup>-5</sup> <sup>[[#References|4]]</sup><br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P3<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| semantic containment<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| genetic failure<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| HGT<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| estimation*<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P4<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| population suicide<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| no surrounding cells with enough toxin<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| no death<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| based on the cell density and the expectation number of surrounding non-mutant<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P5<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| toxin production<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| genetic failure<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| no DNA degradation (w/o antitoxin), no death (with antitoxin)<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| estimation<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P6<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| mutant fitness<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| beneficial mutation<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| outcompetition of the mutant<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| estimation<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P7<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| delay system<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| genetic failure<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| no antitoxin degradation<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| estimation<br />
<br />
|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P8<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| restriction enzyme<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| genetic failure<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| no antitoxin degradation<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| estimation<br />
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|-<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| P9<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| restriction site<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| genetic failure<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| no antitoxin degradation<br />
| style="border:0.035cm solid #000000;padding:0.176cm;"| estimation<br />
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|}<br />
<br />
====Physical containment failure====<br />
<br />
From the experiment we get the escape rate of the cells from the alginate beads is (escaping cells/total cells) is 10<sup>-5</sup> per 12 hours. Assuming 30 minutes of division time, we have 24 generations, and the escape rate per generation is 4 x 10<sup>-6</sup>.<br />
<br />
====Genetic failure====<br />
<br />
There are different processes that lead to a loss of function of a gene<br />
<br />
* Mutation<br />
* Recombination<br />
* Plasmid loss<br />
<br />
'''Losing function because of mutation'''<br />
<br />
Estimating the mutation rate which cause failure of the safety containment of the device<br />
<br />
* Spontaneous mutation rate of a wild-type E coli strain growing on research medium is 3.3 x 10<sup>-9</sup> per nucleotide per generation <sup>[[#References|5]]</sup> <br />
* approximately ⅔ of nucleotide mutation will change amino acid (64 combination of nucleotides code only 20 amino acids)<br />
* approximately 70% of amino acid change will lose the part function <sup>[[#References|6]]</sup><br />
* mean length of genetic coding sequence for E coli (procaryotes) is 924 bp <sup>[[#References|7]]</sup> ≈ 1000 bp <br />
<br />
So the loss function rate of a gene per generation because of mutation is <br />
<br />
P<sub>L</sub><nowiki>= 3.3 x 10</nowiki><sup>-9</sup> x ⅔ x 0.70 x 1000 = 1.54 x 10<sup>-6</sup> <br />
<br />
'''Recombination'''<br />
<br />
Finding a definite rate of recombination is not easy. Guttman and Dykhuizen in 1994 <sup>[[#References|8]]</sup> stated that recombination is believed to occur at such a low frequency, but their experiment showed that the rate could be as high as mutation rate (50 fold higher compared to the mutation rate believed at that moment, but actually it is 5.0 x 10<sup>-9</sup> changes per nucleotide per generation, a bit more than one fold to the value believed now). <br />
<br />
Redoing the previous calculation with this number will lead us to 2.33 x 10<sup>-6</sup> per gene per generation.<br />
<br />
'''Plasmid loss'''<br />
<br />
According to L Boe <sup>[[#References|9]]</sup> plasmid loss rate is defined as the probability that a division of a plasmid-carrying individual results in the birth of one plasmid-free and one plasmid-carrying daughter cell. For the low copy plasmid this rate is about ~1% /division and for high copy plasmid is ~0.01% /division, neglecting the viable/non-viable factor of the cells of losing the plasmid (no selection). If a plasmid with a lost rate of 0.05 is stabilized with a 100% efficient postsegragational killing system, the 5% of the division will result in non viable daughter cell, and we can not see the effect except of increasing the appearing division time by ln 2/ln (1-0.05) = 1.04. Thus in our system we can neglect this plasmid loss assuming that we have antibiotic selection in the beads.<br />
<br />
Assuming only mutation and recombination could happen to delete a function of a gene, we have the total genetic failure rate around 4 x 10<sup>-6</sup> per gene per generation.<br />
<br />
====Total failure probability====<br />
<br />
Having Ptotal = P1.P2.P3.P4.P5 + P1.P6.(P5+P7+P8+P9) and put 1 for unknown probability (P4 and P6) as a worst-case scenario, we come to the failure rate number 6.4 x 10<sup>-11</sup> per generation.<br />
<br />
A volume of one alginate bead is approximately 20 uL. So if the cells grow until they reach the stationary phase (4 x 10<sup>9</sup> cells/ml), we will have 8 x 10<sup>7</sup> cells. If we started from 100 cells per bead, the number of generations we have is <sup>2</sup>log (8 x 10<sup>7</sup> / 100) =19.6 generations. Therefore the total failure becomes 6.4 x 10<sup>-11</sup> x 19.6 = 1.25 x 10<sup>-9</sup>.<br />
<br />
With this method we can also see that the sense-HGT rate is much lower (6.4 x 10<sup>-22</sup>) , almost negligible compared to the outcompetition rate, because it has more back up/redundancy components.<br />
<br />
== Discussion ==<br />
<br />
We have proposed and done an example of assessing biosafety adapting existing method from safety engineering. However it is very clear that the prediction is based on rough estimation and we need more experimental data to verify this estimation. For an example, Torres et al <sup>[[#References|10]]</sup> tested a dual containment killing system and compared it with single containment system. They tested EcoRI based system and Colicin E3-based system and get the efficacy (based on the numbers of successful transformants) each 10<sup>4</sup> and 10<sup>5</sup> respectively but when they combine both system they only got efficacy 10<sup>6</sup>, while theoretically they could reach up to 10<sup>9</sup>. This could give us an idea that predicting a performance of a combination of genetic parts is not as easy as combining the performance of each genetic part. However, we can still conclude that having redundant parts is necessary to reduce the failure rate and make it as low as possible.<br />
<br />
Research in measuring the evolutionary stability in E coli was performed by Sean C Sleight et al <sup>[[#References|11]]</sup> where they measured it of BioBrick-assembled genetic circuits in E coli over multiple generation by measuring the number of loss-functioning mutants. They concluded that to make a robust GE bacteria, one has to take into account 3 principles:<br />
# High expression of genetic circuits comes with the cost of low evolutionary stability (for example induced vs non-induced system)<br />
#Avoid repeated sequences because it will more likely be mutated<br />
#Use of inducible promoters generally increases evolutionary stability compared to constitutive promoters<br />
<br />
To predict the robustness of the system from mutation, we have to take into account for how long time (i.e. how many generations) we want our system to work. From their experiment for example, the AHL induced TetR-GFP cells lose their functions after 30 generations, while the uninduced ones lose their function slower (50% after 300 generations). A similar idea is also shown from our work where the mutation rate we got from literature research is in the unit of per generation. This is how reproduction differs biological system from other engineering systems. In engineering we also have failure probability as a function of time because of the life cycle of the system, but the system is not reproducing so it has no variability. <br />
<br />
Analyzing key elements that cause failure in any systems including synthetic biology systems is essential for system improvement and prediction of the failure. Adaptation of classical safety engineering methods needs to take into account the uniqueness properties of synthetic biology. Reproducibility and complexity are two example of areas that need to be studied deeper in this context. We invite future iGEM teams to use and improve our assessment framework, build a solid assessment protocol towards a better understanding of biosafety and successful application of synthetic biology.<br />
<br />
== Conclusion ==<br />
*We can adapt methods in safety engineering to assess risk in biosafety<br />
*We need more experimental data to confirm the risk assessing method<br />
<br />
== Perspectives ==<br />
<br />
*We need a method to predict the evolutionary stability of circuits from the properties of their parts, but the emergent behaviours of circuits will likely make prediction difficult. Thus it will be very useful if each BioBricks parts is completed with evolutionary stability data sheet, so we can make prediction of the stability of more complex circuits.<br />
*This safety assessment method is useful for identifying key elements of having failure in synthetic circuit systems before we go further to the real application. Thus we recommend for the future iGEM team to use our assessment method and give feedback to the community by improving this framework.<br />
<br />
==References==<br />
[[#Background|1]] - Genya V Dana et al. Four steps to avoid a synthetic biology disaster. 2012. Nature vol 483<br />
<br />
[[#Methods_and_Results|2]] - WorkSafe Victoria. A handbook for workplaces, controlling OHS hazards and risks. 2007. Edition No.1<br />
<br />
[[#Methods_and_Results|3]] - “Five steps to risk assessment” from [http://www.hse.gov.uk/risk/fivesteps.htm http://www.hse.gov.uk/risk/fivesteps.htm]. - risk management method in workplaces complying with health and safety law <br />
<br />
[[#Check_controls|4]] - Soren J Sorensen et al. 2005. Studying plasmid horizontal gene transfer in situ: a critical review. Nature Reviews Microbiology Vol 3<br />
<br />
[[#Check_controls|5]] - Heewook Lee et al., Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing. 2012. PNAS E2774-E2783<br />
<br />
[[#Check_controls|6]] - Stanley A. Sawyer et al. Prevalence of positive selection among nearly neutral amino acid replacements in Drosophila. 2007. PNAS 104(16):6504-6510<br />
<br />
[[#Check_controls|7]] - Lin Xu et al. Average gene length is highly conserved in prokaryotes and eukaryotes and diverges only between two kingdoms. 2006. Mol. Biol. Evol. 23(6):1107-1108<br />
<br />
[[#Check_controls|8]] - David S Guttmand and Daniel E. Dykhuizen. 1994. Clonal divergence in Escherichia coli as a result of recombination, not mutation. Science Vol 266<br />
<br />
[[#Check_controls|9]] - L.Boe. 1996. Estimation of Plasmid Loss Rates in Bacterial Populations with a Reference to the Reproducibility of Stability Experiments. Plasmid 36, 161–167. Article No. 0043<br />
<br />
[[#Discussion|10]] - Torres B et al. 2003. A dual lethal system to enhance containment of recombinant micro-organisms. Microbiology. 2003 Dec;149(Pt 12):3595-601.<br />
<br />
[[#Discussion|11]] - Sean C Sleight et al., Designing and engineering evolutionary robust genetic circuits. 2010. Journal of Biological Engineering, 4:12<br />
</div><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/File:ParisB_FTA.pngFile:ParisB FTA.png2012-10-26T23:12:46Z<p>Aleksandra: uploaded a new version of &quot;File:ParisB FTA.png&quot;</p>
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<div></div>Aleksandrahttp://2012.igem.org/File:ParisB_FTA.pngFile:ParisB FTA.png2012-10-26T23:07:52Z<p>Aleksandra: uploaded a new version of &quot;File:ParisB FTA.png&quot;</p>
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<div></div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/perceptionTeam:Paris Bettencourt/Human Practice/perception2012-10-26T22:57:17Z<p>Aleksandra: </p>
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<div id="grouptitle">Team aWAREness </div><br />
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During this summer, all of us gained knowledge in synthetic biology and learned lab skills, but that wasn't all. <br />
From the beginning of our brainstorming sessions, safety questions came up in our discussions. Our mutual interest in this topic lead us to center our project on safeguard systems and human practices related to public awareness and risk assesssment. This meant that we had to work hard not only on our wet lab project, but also on human practices.<br />
To our delight, this effort resulted not only in community outreach, but also changed our own opinion on biosafety in the context of synthetic biology. We feel that our Human Practice project changed each and every one of us. Here are our personal perceptions.<br />
<br />
[[Image:Julianne.png|thumb|left|100px]] When we first began our project, I was really skeptical about the long term goals of releasing bacteria into the environment. However, during the debate we held some members of the "government", arguing in favor of the release of genetically modified bacteria, reminded the audience that there was a time when some held the opinion that airplanes were infernal machines that would only end in doom. We were questioned by judges in Amsterdam, who said that bacterial containment is impossible. I was inspired by the debaters. We have two options, we can either accept that biological containment is impossible, or we can try to study this problem and develop containment devices. In the end we may come to the conclusion that the risks are too great to ever release GE bacteria into the environment, but if we do not try to explore this problem we will do a great disservice to all the beautiful and brilliant iGEM projects dedicated to bioremediation. <br />
<br />
<br />
<br />
[[Image:Ernest.png|thumb|left|100px]] Human practices definitely brought a new dimension to our project. The question of biosafety is too broad to be tackled from the "narrow" point of view of a pure synthetic biologist, and we realized how important the contact and the discussion with the population is when dealing with such a sensitive topic. In our case, both the risks and the potential benefits are huge, leading to very polarized opinions among the interviewees. I strongly believe that scientists must make an effort to increase the transparency of their results and not push their ideas if the population doesn't accept them, in order to reduce the gap that now separates them from the rest of the population.<br />
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<br />
[[Image:Aishah.png|thumb|left|100px]] The most remarkable thing I learned from our human practice project is the level of public awareness on genetic modification. I am not a biologist myself, and before I did not really care about this field--I always thought 'the experts knows better'. But after seeing the debate and even the discussion among high school students about this field, I was surprised with their opinion showing how much they actually aware. I guess it may be a bit related with the different education culture in France and in my country where the students are less encouraged to speak about their thought; but as a prospective scientist I learned that I should care more about public opinion, as well as expose myself with new knowledge and information.<br />
<br />
<br />
<br />
<br />
[[Image:Dylan.png|thumb|left|100px]] Our project was initially based upon the idea of genetically modified bacteria that could be sprayed along with DDT. The bacteria would degrade the DDT after some time, hopefully having a less drastic environmental impact, as the DDT would soon disappear. We started to consider consider human practices as an important issue and realized that the DDT project could have other unaddressed dangers. What if the DDT degrading genes were transferred to other species? DDT degrading gene could be transferred to mosquitoes, and then our system would have done something far, far worse than the its potential for good. But I know that there must be worthy risks in terms of environmental applications of genetically modified bacteria. Through human practices I learned that most people would agree, through a proper weighing of benefit vs. risk, certain projects should be applied in the environment as long as we use proper genetic safeguards for safety.<br />
<br />
<br />
[[Image:Claire.png|thumb|left|100px]] I could not imagine how anyone in his/her right mind could be opposed to synthetic biology and its applications. I was convinced that if people were, it was because they did not really know much about the field, because they are ignorant. Therefore if we educated them, they would realize how GREAT SB is and would accept it.<br />
Now, thanks to the human practice project, I realized that this naïve vision of things is completely false and also very dangerous!<br />
First realization: People have the legitimate right to be opposed to synthetic biology. There is no link between ignorance of SB and rejection of its applications<br />
Second realization: Every citizen should have a say in what technologies they want or do not want. Experts should not be the ones making the final call!<br />
Third realization: Education is very important. The aim should be to give people all the necessary tools to understand what exactly is going on, and so that they can therefore discuss in the most illuminated way possible if they want or not the technology as part of their world (education’s aim should absolutely not be making people agree with us and accept synthetic biology! This vision is dangerous!!!)<br />
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[[Image:Denis.png|thumb|left|100px]] I came from Physics and until last year I didn't know anything about synthetic biology and biodegradation. However, I was always interested in projects intended to save the world. Or, at least, how to deal with problems caused by humanity? Due to that, I always was concerned about the big amount of waste produced by peoples. After I learned that Synthetic biology develops methods to solve those problems, I came up with the idea to degrade insecticide using bacteria, but with a delay: first, to kill insects, and after some delay, to degrade insecticide to avoid side effects. At that time, I had no idea about gene transfer, and that scientists don't release any synthetic bacteria to the environment. For me it was really surprising! How we could benefit from such great ideas like iGEM projects without having any possibility to use bacteria outside the lab? A lot of question appeared. Is it possible to create a safe containment system? What is the risk? Would ordinary citizens be interested in such projects? Those questions gave rise to our iGEM project, and human practice in parallel with theoretical and laboratory work partially gave me an answer to it.<br />
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<br />
[[Image:Jean.png|thumb|left|100px]] What I found good from the Human practice part, was the interview we had with specialist, which was very interesting, because we could have had different point of views, and in the same time some really good and rich discussions. Also the report was good for me to keep a trace of the historical events that drive us in our situation. The debate was a good idea and we couldn't have expected more from it.<br />
Concerning teaching to the high school student synthetic biology, it's very disturbing for me, because in one hand, biotech companies give tools to high schools to build transgenic crops, in order to make their reputation better and not in a total altruistic way. In the other hand, we suggest to teach synthetic biology to kids, and for me it's hard to know whether it's really to teach them how to be critical toward this technology, or in fact doing the same as biotech companies, because they're still young and most of them won't see limits, even if they are taught.<br />
I think that at least, it should be taught in university for biologist, which is not done so far, unless being in a synthetic biology curriculum.<br />
<br />
[[Image:Guillaume.png|thumb|left|100px]] We started to consider human practices as an important issue since the beginning of the project. Indeed we show that a lot of previous iGEM project and our project first ideas had the goal to be released in nature but none of them had a serious safety device. During human practice I realize that zero risk doesn’t exist and nevertheless we can use genetically engineered organism for specific usage and assets risk for this specific usage as long as we discuss it with a large population. I learn that most of the people would agree that certain projects should be applied in the environment as long as we use safety devices.<br />
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<br />
<br />
<br />
[[Image:Zoran.png|thumb|left|100px]] I always thought the public opinion on genetically modified organisms was based on fear and ignorance which led to the irrational behaviour and irrational arguments. This bothered me and still bothers me very much because it is completely opposite to how I deal with problems I encounter. We should try to make as much informed and educated opinion as we can and by doing this try to overcome our fears, often based on our wild imagination triggered by people trying to exploit this basic human emotion. Throughout our intensive work on issues of human practices my opinion about public changed resulting in more understanding and concern from my side. I was glad to hear that the public is willing to accept risks if they see considerable benefits. But my main realization, selfish in a way, was that I as a synthetic biologist won’t be able to change the world if I don’t make public feel safer by doing what I do the best – science. That’s why I accepted to do an iGEM project which deals with biosafety from a scientific point of view. Synthetic biologists should not contain themselves to only living in the lab, in an ivory tower detached from the public, but rather collaborate with the public to reach the best decisions for community. In a sense, we should shift from making change only in the lab to making change in the whole world.<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/OverviewTeam:Paris Bettencourt/Overview2012-10-26T22:52:37Z<p>Aleksandra: /* Evaluation */</p>
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1b/MOUSECURSORPB12.png" width="60px" border="0" /><span></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/e/e3/PhysicalContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/f/f6/PhysicalContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV"><b>1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth</b> </div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/0/09/SemanticContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br><b>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. </b></div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
<b>3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.</b><br />
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1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
<b>4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.</b></div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/d/da/SkullIcon.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.<br><br><b>5) Suicide system: Once the anti-toxin concentration is below a given threshold, the toxin is no longer inhibited. It kills the cell as well as its neighbors, and eliminates extracellular DNA via its DNase activity.</b></div></span></a><br />
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==Our project - a hypothetical case study==<br />
<br />
Imagine a farmer that wants to measure the nutrient concentration in her field, in order to optimize her fertilizer use. We could provide her with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in alginate beads. She would spread the beads in her field, wait for 12 hours, and then check if they are glowing in response to the nitrates in the soil.<br />
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We want this system to work the way the original designers intended. We also want to reduce the chance that the engineered bacteria will survive in the soil, release intact DNA, or transfer genes to a soil microbe.<br />
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Our system will be implemented as a pair of plasmids, compatible with the most commonly used BioBrick plasmids, and therefore easily integrated with existing systems. The AgrEColi are now running bWARE, and express a new containment functionality. <br />
<br />
Shortly after the beads enter the soil, the delay system triggers and the DNA-degrading colicin proteins become active. The colicins specifically recognize and enter E. coli cells, killing them by completely degrading their genomes. The colicins also degrade DNA loose in the environment, without harming the native bacterial species.<br />
<br />
Every field is a different ecosystem with a different composition of native species. The potential consequences of horizontal gene transfer are therefore difficult to predict in general. By destroying the information contained in DNA, our system reduces the chance that introduced DNA will replicate in the new environment.<br />
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==Objectives==<br />
<br />
Our project aims to:<br />
<br />
*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications, and perform a fault tree analysis for our project as an example<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
<br />
To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
<br />
<table id="tableboxed" style="border-color:rgb(176,18,31);"><br />
<tr><br />
<td> <br />
====Key Safety Device Design Features====<br />
<br />
*The physical capsule prevents Genetically Engineered Bacteria (GEB) from escaping into the environment.<br />
*The DNAse toxin kills cells and destroys genetic information.<br />
*A population-level mechanism compensates system failure in single cells.<br />
*Plasmid destruction with restriction enzymes is an irreversible trigger.<br />
*A specific toxin targeting mechanism reduces system impact on native fauna.<br />
*In case HGT occurs, semantic containment prevents the expression of genes from the GEB.<br />
*The modular design supports integration with existing iGEM projects.<br />
*The use of several redundant modules compensates the failure of one of them.<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
==Evaluation==<br />
<div id="boston"><br />
<br />
<br />
It is essential not only to implement a safety system, but also to evaluate its efficiency.<br />
We have addressed this issue by creating a fault tree analysis for our system, but we believe that such approach should be recommended for containment system. You can find more about it on our [https://2012.igem.org/Team:Paris_Bettencourt/Modeling Safety Assessment page].<br />
<br />
<center>[[Image:ParisB_FTA.png]]</center><br />
</div><br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/OverviewTeam:Paris Bettencourt/Overview2012-10-26T22:51:50Z<p>Aleksandra: /* Evaluation */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<br />
<div id="grouptitle">Project Overview</div><br />
<br><br />
<html><br />
<style type="text/css"><br />
.gallerycontainer{<br />
position: relative;<br />
/*Add a height attribute and set to largest image's height to prevent overlaying*/<br />
}<br />
<br />
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border: 1px solid white;<br />
margin: 0 5px 5px 0;<br />
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left: 598px;<br />
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top: -555px;<br />
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background-color: transparent;<br />
}<br />
/*<br />
.thumbnail:hover img{<br />
border: 1px solid blue;<br />
}<br />
*/<br />
.thumbnail span { /*CSS for enlarged image*/<br />
position: absolute;<br />
top : 80px;<br />
border: none;<br />
visibility: hidden;<br />
color: black;<br />
text-decoration: none;<br />
}<br />
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border-width: 0;<br />
padding: 2px;<br />
}<br />
<br />
.thumbnail:hover span{ /*CSS for enlarged image*/<br />
visibility: visible;<br />
top: 60 px;<br />
left: -70px; /*position where enlarged image should offset horizontally */<br />
z-index: 50;<br />
}<br />
<br />
<br />
</style><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/18/TotalBiosafety.png" width="500px" style="left: 32px;position: absolute;top: 443px;"/><br />
<br />
<div class="gallerycontainer"><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1b/MOUSECURSORPB12.png" width="60px" border="0" /><span></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/e/e3/PhysicalContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/f/f6/PhysicalContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV"><b>1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth</b> </div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/0/09/SemanticContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br><b>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. </b></div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
<b>3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.</b><br />
</div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/5/5c/RestrictionSystem.png" width="60px" border="0" /><span><img src="/wiki/images/9/97/Restriction2PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
<b>4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.</b></div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/d/da/SkullIcon.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.<br><br><b>5) Suicide system: Once the anti-toxin concentration is below a given threshold, the toxin is no longer inhibited. It kills the cell as well as its neighbors, and eliminates extracellular DNA via its DNase activity.</b></div></span></a><br />
<br />
<br />
<br />
</div><br />
</html><br />
<br />
<!-- ########## Don't edit above ########## --><br />
<br><br />
<br><br />
<br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
==Our project - a hypothetical case study==<br />
<br />
Imagine a farmer that wants to measure the nutrient concentration in her field, in order to optimize her fertilizer use. We could provide her with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in alginate beads. She would spread the beads in her field, wait for 12 hours, and then check if they are glowing in response to the nitrates in the soil.<br />
<br />
We want this system to work the way the original designers intended. We also want to reduce the chance that the engineered bacteria will survive in the soil, release intact DNA, or transfer genes to a soil microbe.<br />
<br />
Our system will be implemented as a pair of plasmids, compatible with the most commonly used BioBrick plasmids, and therefore easily integrated with existing systems. The AgrEColi are now running bWARE, and express a new containment functionality. <br />
<br />
Shortly after the beads enter the soil, the delay system triggers and the DNA-degrading colicin proteins become active. The colicins specifically recognize and enter E. coli cells, killing them by completely degrading their genomes. The colicins also degrade DNA loose in the environment, without harming the native bacterial species.<br />
<br />
Every field is a different ecosystem with a different composition of native species. The potential consequences of horizontal gene transfer are therefore difficult to predict in general. By destroying the information contained in DNA, our system reduces the chance that introduced DNA will replicate in the new environment.<br />
<br />
==Objectives==<br />
<br />
Our project aims to:<br />
<br />
*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications, and perform a fault tree analysis for our project as an example<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
<br />
To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
<br />
<table id="tableboxed" style="border-color:rgb(176,18,31);"><br />
<tr><br />
<td> <br />
====Key Safety Device Design Features====<br />
<br />
*The physical capsule prevents Genetically Engineered Bacteria (GEB) from escaping into the environment.<br />
*The DNAse toxin kills cells and destroys genetic information.<br />
*A population-level mechanism compensates system failure in single cells.<br />
*Plasmid destruction with restriction enzymes is an irreversible trigger.<br />
*A specific toxin targeting mechanism reduces system impact on native fauna.<br />
*In case HGT occurs, semantic containment prevents the expression of genes from the GEB.<br />
*The modular design supports integration with existing iGEM projects.<br />
*The use of several redundant modules compensates the failure of one of them.<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
==Evaluation==<br />
<div id="boston"><br />
<br />
<br />
It is essential not only to implement a safety system, but also to evaluate its efficiency.<br />
We have addressed this issue by creating a fault tree analysis for our system, but we believe that such approach should be recommended for containment system. You can find more about it on our [https://2012.igem.org/Team:Paris_Bettencourt/Modeling Safety Assessment page].<br />
<br />
<center>[[Image:ParisB_FTA.png]]</center><br />
<br />
<br />
<div id="boston"><br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/OverviewTeam:Paris Bettencourt/Overview2012-10-26T22:50:47Z<p>Aleksandra: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<br />
<div id="grouptitle">Project Overview</div><br />
<br><br />
<html><br />
<style type="text/css"><br />
.gallerycontainer{<br />
position: relative;<br />
/*Add a height attribute and set to largest image's height to prevent overlaying*/<br />
}<br />
<br />
.thumbnail img {<br />
border: 1px solid white;<br />
margin: 0 5px 5px 0;<br />
position: relative;<br />
top: -50px;<br />
left: 50px;<br />
}<br />
#txtOV {<br />
left: 598px;<br />
position: relative;<br />
top: -555px;<br />
width: 400px;<br />
}<br />
<br />
.thumbnail:hover{<br />
background-color: transparent;<br />
}<br />
/*<br />
.thumbnail:hover img{<br />
border: 1px solid blue;<br />
}<br />
*/<br />
.thumbnail span { /*CSS for enlarged image*/<br />
position: absolute;<br />
top : 80px;<br />
border: none;<br />
visibility: hidden;<br />
color: black;<br />
text-decoration: none;<br />
}<br />
<br />
.thumbnail span img{ /*CSS for enlarged image*/<br />
border-width: 0;<br />
padding: 2px;<br />
}<br />
<br />
.thumbnail:hover span{ /*CSS for enlarged image*/<br />
visibility: visible;<br />
top: 60 px;<br />
left: -70px; /*position where enlarged image should offset horizontally */<br />
z-index: 50;<br />
}<br />
<br />
<br />
</style><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/18/TotalBiosafety.png" width="500px" style="left: 32px;position: absolute;top: 443px;"/><br />
<br />
<div class="gallerycontainer"><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1b/MOUSECURSORPB12.png" width="60px" border="0" /><span></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/e/e3/PhysicalContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/f/f6/PhysicalContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV"><b>1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth</b> </div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/0/09/SemanticContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br><b>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. </b></div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
<b>3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.</b><br />
</div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/5/5c/RestrictionSystem.png" width="60px" border="0" /><span><img src="/wiki/images/9/97/Restriction2PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
<b>4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.</b></div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/d/da/SkullIcon.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.<br><br><b>5) Suicide system: Once the anti-toxin concentration is below a given threshold, the toxin is no longer inhibited. It kills the cell as well as its neighbors, and eliminates extracellular DNA via its DNase activity.</b></div></span></a><br />
<br />
<br />
<br />
</div><br />
</html><br />
<br />
<!-- ########## Don't edit above ########## --><br />
<br><br />
<br><br />
<br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
==Our project - a hypothetical case study==<br />
<br />
Imagine a farmer that wants to measure the nutrient concentration in her field, in order to optimize her fertilizer use. We could provide her with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in alginate beads. She would spread the beads in her field, wait for 12 hours, and then check if they are glowing in response to the nitrates in the soil.<br />
<br />
We want this system to work the way the original designers intended. We also want to reduce the chance that the engineered bacteria will survive in the soil, release intact DNA, or transfer genes to a soil microbe.<br />
<br />
Our system will be implemented as a pair of plasmids, compatible with the most commonly used BioBrick plasmids, and therefore easily integrated with existing systems. The AgrEColi are now running bWARE, and express a new containment functionality. <br />
<br />
Shortly after the beads enter the soil, the delay system triggers and the DNA-degrading colicin proteins become active. The colicins specifically recognize and enter E. coli cells, killing them by completely degrading their genomes. The colicins also degrade DNA loose in the environment, without harming the native bacterial species.<br />
<br />
Every field is a different ecosystem with a different composition of native species. The potential consequences of horizontal gene transfer are therefore difficult to predict in general. By destroying the information contained in DNA, our system reduces the chance that introduced DNA will replicate in the new environment.<br />
<br />
==Objectives==<br />
<br />
Our project aims to:<br />
<br />
*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications, and perform a fault tree analysis for our project as an example<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
<br />
To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
<br />
<table id="tableboxed" style="border-color:rgb(176,18,31);"><br />
<tr><br />
<td> <br />
====Key Safety Device Design Features====<br />
<br />
*The physical capsule prevents Genetically Engineered Bacteria (GEB) from escaping into the environment.<br />
*The DNAse toxin kills cells and destroys genetic information.<br />
*A population-level mechanism compensates system failure in single cells.<br />
*Plasmid destruction with restriction enzymes is an irreversible trigger.<br />
*A specific toxin targeting mechanism reduces system impact on native fauna.<br />
*In case HGT occurs, semantic containment prevents the expression of genes from the GEB.<br />
*The modular design supports integration with existing iGEM projects.<br />
*The use of several redundant modules compensates the failure of one of them.<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
==Evaluation==<br />
It is essential not only to implement a safety system, but also to evaluate its efficiency.<br />
We have addressed this issue by creating a fault tree analysis for our system, but we believe that such approach should be recommended for containment system. You can find more about it on our [https://2012.igem.org/Team:Paris_Bettencourt/Modeling Safety Assessment page].<br />
<br />
<center>[[Image:ParisB_FTA.png]]</center><br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/OverviewTeam:Paris Bettencourt/Overview2012-10-26T22:38:19Z<p>Aleksandra: /* Key Killswitch Design Features */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<br />
<div id="grouptitle">Project Overview</div><br />
<br><br />
<html><br />
<style type="text/css"><br />
.gallerycontainer{<br />
position: relative;<br />
/*Add a height attribute and set to largest image's height to prevent overlaying*/<br />
}<br />
<br />
.thumbnail img {<br />
border: 1px solid white;<br />
margin: 0 5px 5px 0;<br />
position: relative;<br />
top: -50px;<br />
left: 50px;<br />
}<br />
#txtOV {<br />
left: 598px;<br />
position: relative;<br />
top: -555px;<br />
width: 400px;<br />
}<br />
<br />
.thumbnail:hover{<br />
background-color: transparent;<br />
}<br />
/*<br />
.thumbnail:hover img{<br />
border: 1px solid blue;<br />
}<br />
*/<br />
.thumbnail span { /*CSS for enlarged image*/<br />
position: absolute;<br />
top : 80px;<br />
border: none;<br />
visibility: hidden;<br />
color: black;<br />
text-decoration: none;<br />
}<br />
<br />
.thumbnail span img{ /*CSS for enlarged image*/<br />
border-width: 0;<br />
padding: 2px;<br />
}<br />
<br />
.thumbnail:hover span{ /*CSS for enlarged image*/<br />
visibility: visible;<br />
top: 60 px;<br />
left: -70px; /*position where enlarged image should offset horizontally */<br />
z-index: 50;<br />
}<br />
<br />
<br />
</style><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/18/TotalBiosafety.png" width="500px" style="left: 32px;position: absolute;top: 443px;"/><br />
<br />
<div class="gallerycontainer"><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1b/MOUSECURSORPB12.png" width="60px" border="0" /><span></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/e/e3/PhysicalContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/f/f6/PhysicalContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV"><b>1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth</b> </div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/0/09/SemanticContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br><b>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. </b></div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
<b>3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.</b><br />
</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/5/5c/RestrictionSystem.png" width="60px" border="0" /><span><img src="/wiki/images/9/97/Restriction2PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
<b>4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.</b></div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/d/da/SkullIcon.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.<br><br><b>5) Suicide system: Once the anti-toxin concentration is below a given threshold, the toxin is no longer inhibited. It kills the cell as well as its neighbors, and eliminates extracellular DNA via its DNase activity.</b></div></span></a><br />
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==Our project - a hypothetical case study==<br />
<br />
Imagine a farmer that wants to measure the nutrient concentration in her field, in order to optimize her fertilizer use. We could provide her with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in alginate beads. She would spread the beads in her field, wait for 12 hours, and then check if they are glowing in response to the nitrates in the soil.<br />
<br />
We want this system to work the way the original designers intended. We also want to reduce the chance that the engineered bacteria will survive in the soil, release intact DNA, or transfer genes to a soil microbe.<br />
<br />
Our system will be implemented as a pair of plasmids, compatible with the most commonly used BioBrick plasmids, and therefore easily integrated with existing systems. The AgrEColi are now running bWARE, and express a new containment functionality. <br />
<br />
Shortly after the beads enter the soil, the delay system triggers and the DNA-degrading colicin proteins become active. The colicins specifically recognize and enter E. coli cells, killing them by completely degrading their genomes. The colicins also degrade DNA loose in the environment, without harming the native bacterial species.<br />
<br />
Every field is a different ecosystem with a different composition of native species. The potential consequences of horizontal gene transfer are therefore difficult to predict in general. By destroying the information contained in DNA, our system reduces the chance that introduced DNA will replicate in the new environment.<br />
<br />
==Objectives==<br />
<br />
Our project aims to:<br />
<br />
*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications, and perform a fault tree analysis for our project as an example<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
<br />
To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
<br />
<table id="tableboxed" style="border-color:rgb(176,18,31);"><br />
<tr><br />
<td> <br />
====Key Safety Device Design Features====<br />
<br />
*The physical capsule prevents Genetically Engineered Bacteria (GEB) from escaping into the environment.<br />
*The DNAse toxin kills cells and destroys genetic information.<br />
*A population-level mechanism compensates system failure in single cells.<br />
*Plasmid destruction with restriction enzymes is an irreversible trigger.<br />
*A specific toxin targeting mechanism reduces system impact on native fauna.<br />
*In case HGT occurs, semantic containment prevents the expression of genes from the GEB.<br />
*The modular design supports integration with existing iGEM projects.<br />
*The use of several redundant modules compensates the failure of one of them.<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/OverviewTeam:Paris Bettencourt/Overview2012-10-26T22:31:28Z<p>Aleksandra: /* Objectives */</p>
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<div id="grouptitle">Project Overview</div><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1b/MOUSECURSORPB12.png" width="60px" border="0" /><span></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/e/e3/PhysicalContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/f/f6/PhysicalContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV"><b>1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth</b> </div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/0/09/SemanticContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br><b>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. </b></div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
<b>3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.</b><br />
</div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/5/5c/RestrictionSystem.png" width="60px" border="0" /><span><img src="/wiki/images/9/97/Restriction2PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
<b>4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.</b></div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/d/da/SkullIcon.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.<br><br><b>5) Suicide system: Once the anti-toxin concentration is below a given threshold, the toxin is no longer inhibited. It kills the cell as well as its neighbors, and eliminates extracellular DNA via its DNase activity.</b></div></span></a><br />
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<br />
==Our project - a hypothetical case study==<br />
<br />
Imagine a farmer that wants to measure the nutrient concentration in her field, in order to optimize her fertilizer use. We could provide her with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in alginate beads. She would spread the beads in her field, wait for 12 hours, and then check if they are glowing in response to the nitrates in the soil.<br />
<br />
We want this system to work the way the original designers intended. We also want to reduce the chance that the engineered bacteria will survive in the soil, release intact DNA, or transfer genes to a soil microbe.<br />
<br />
Our system will be implemented as a pair of plasmids, compatible with the most commonly used BioBrick plasmids, and therefore easily integrated with existing systems. The AgrEColi are now running bWARE, and express a new containment functionality. <br />
<br />
Shortly after the beads enter the soil, the delay system triggers and the DNA-degrading colicin proteins become active. The colicins specifically recognize and enter E. coli cells, killing them by completely degrading their genomes. The colicins also degrade DNA loose in the environment, without harming the native bacterial species.<br />
<br />
Every field is a different ecosystem with a different composition of native species. The potential consequences of horizontal gene transfer are therefore difficult to predict in general. By destroying the information contained in DNA, our system reduces the chance that introduced DNA will replicate in the new environment.<br />
<br />
==Objectives==<br />
<br />
Our project aims to:<br />
<br />
*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications, and perform a fault tree analysis for our project as an example<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
<br />
To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
<br />
<table id="tableboxed" style="border-color:rgb(176,18,31);"><br />
<tr><br />
<td> <br />
====Key Killswitch Design Features====<br />
<br />
*The DNAse toxin kills cells and destroys genetic information.<br />
*A population-level mechanism compensates system failure in single cells.<br />
*Plasmid destruction with restriction enzymes is an irreversible trigger.<br />
*A specific toxin targeting mechanism reduces system impact on native fauna.<br />
*The modular design supports integration with existing iGEM projects.<br />
*The use of several redundant modules compensates the failure of one of them.<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/perceptionTeam:Paris Bettencourt/Human Practice/perception2012-10-26T22:27:09Z<p>Aleksandra: </p>
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<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="boston"><br />
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<div id="grouptitle">Team aWAREness </div><br />
<br />
During this summer, all of us gained knowledge in synthetic biology and learned lab skills, but that wasn't all. <br />
From the beginning of our brainstorming sessions, safety questions came up in our discussions. Our mutual interest in this topic lead us to center our project on safeguard systems and human practices related to public awareness and risk assesssment. This meant that we had to work hard not only on our wet lab project, but also on human practices.<br />
To our delight, this effort resulted not only in community outreach, but also changed our own opinion on biosafety in the context of synthetic biology. We feel that our Human Practice project changed each and every one of us. Here are our personal perceptions.<br />
<br />
[[Image:Julianne.png|thumb|left|100px]] When we first began our project, I was really skeptical about the long term goals of releasing bacteria into the environment. However, during the debate we held some members of the "government", arguing in favor of the release of genetically modified bacteria, reminded the audience that there was a time when some held the opinion that airplanes were infernal machines that would only end in doom. We were questioned by judges in Amsterdam, who said that bacterial containment is impossible. I was inspired by the debaters. We have two options, we can either accept that biological containment is impossible, or we can try to study this problem and develop containment devices. In the end we may come to the conclusion that the risks are too great to ever release GE bacteria into the environment, but if we do not try to explore this problem we will do a great disservice to all the beautiful and brilliant iGEM projects dedicated to bioremediation. <br />
<br />
<br />
<br />
[[Image:Ernest.png|thumb|left|100px]] Human practices definitely brought a new dimension to our project. The question of biosafety is too broad to be tackled from the "narrow" point of view of a pure synthetic biologist, and we realized how important the contact and the discussion with the population is when dealing with such a sensitive topic. In our case, both the risks and the potential benefits are huge, leading to very polarized opinions among the interviewees. I strongly believe that scientists must make an effort to increase the transparency of their results and not push their ideas if the population doesn't accept them, in order to reduce the gap that now separates them from the rest of the population.<br />
<br />
<br />
[[Image:Aishah.png|thumb|left|100px]] The most remarkable thing I learned from our human practice project is the level of public awareness on genetic modification. I am not a biologist myself, and before I did not really care about this field--I always thought 'the experts knows better'. But after seeing the debate and even the discussion among high school students about this field, I was surprised with their opinion showing how much they actually aware. I guess it may be a bit related with the different education culture in France and in my country where the students are less encouraged to speak about their thought; but as a prospective scientist I learned that I should care more about public opinion, as well as expose myself with new knowledge and information.<br />
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[[Image:Dylan.png|thumb|left|100px]] Our project was initially based upon the idea of genetically modified bacteria that could be sprayed along with DDT. The bacteria would degrade the DDT after some time, hopefully having a less drastic environmental impact, as the DDT would soon disappear. We started to consider consider human practices as an important issue and realized that the DDT project could have other unaddressed dangers. What if the DDT degrading genes were transferred to other species? DDT degrading gene could be transferred to mosquitoes, and then our system would have done something far, far worse than the its potential for good. But I know that there must be worthy risks in terms of environmental applications of genetically modified bacteria. Through human practices I learned that most people would agree, through a proper weighing of benefit vs. risk, certain projects should be applied in the environment as long as we use proper genetic safeguards for safety.<br />
<br />
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[[Image:Claire.png|thumb|left|100px]] I could not imagine how anyone in his/her right mind could be opposed to synthetic biology and its applications. I was convinced that if people were, it was because they did not really know much about the field, because they are ignorant. Therefore if we educated them, they would realize how GREAT SB is and would accept it.<br />
Now, thanks to the human practice project, I realized that this naïve vision of things is completely false and also very dangerous!<br />
First realization: People have the legitimate right to be opposed to synthetic biology. There is no link between ignorance of SB and rejection of its applications<br />
Second realization: Every citizen should have a say in what technologies they want or do not want. Experts should not be the ones making the final call!<br />
Third realization: Education is very important. The aim should be to give people all the necessary tools to understand what exactly is going on, and so that they can therefore discuss in the most illuminated way possible if they want or not the technology as part of their world (education’s aim should absolutely not be making people agree with us and accept synthetic biology! This vision is dangerous!!!)<br />
<br />
[[Image:Denis.png|thumb|left|100px]] I came from Physics and until last year I didn't know anything about synthetic biology and biodegradation. However, I was always interested in projects intended to save the world. Or, at least, how to deal with problems caused by humanity? Due to that, I always was concerned about the big amount of waste produced by peoples. After I learned that Synthetic biology develops methods to solve those problems, I came up with the idea to degrade insecticide using bacteria, but with a delay: first, to kill insects, and after some delay, to degrade insecticide to avoid side effects. At that time, I had no idea about gene transfer, and that scientists don't release any synthetic bacteria to the environment. For me it was really surprising! How we could benefit from such great ideas like iGEM projects without having any possibility to use bacteria outside the lab? A lot of question appeared. Is it possible to create a safe containment system? What is the risk? Would ordinary citizens be interested in such projects? Those questions gave rise to our iGEM project, and human practice in parallel with theoretical and laboratory work partially gave me an answer to it.<br />
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[[Image:Jean.png|thumb|left|100px]] What I found good from the Human practice part, was the interview we had with specialist, which was very interesting, because we could have had different point of views, and in the same time some really good and rich discussions. Also the report was good for me to keep a trace of the historical events that drive us in our situation. The debate was a good idea and we couldn't have expected more from it.<br />
Concerning teaching to the high school student synthetic biology, it's very disturbing for me, because in one hand, biotech companies give tools to high schools to build transgenic crops, in order to make their reputation better and not in a total altruistic way. In the other hand, we suggest to teach synthetic biology to kids, and for me it's hard to know whether it's really to teach them how to be critical toward this technology, or in fact doing the same as biotech companies, because they're still young and most of them won't see limits, even if they are taught.<br />
I think that at least, it should be taught in university for biologist, which is not done so far, unless being in a synthetic biology curriculum.<br />
<br />
[[Image:Guillaume.png|thumb|left|100px]] We started to consider human practices as an important issue since the beginning of the project. Indeed we show that a lot of previous iGEM project and our project first ideas had the goal to be released in nature but none of them had a serious safety device. During human practice I realize that zero risk doesn’t exist and nevertheless we can use genetically engineered organism for specific usage and assets risk for this specific usage as long as we discuss it with a large population. I learn that most of the people would agree that certain projects should be applied in the environment as long as we use safety devices.<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/perceptionTeam:Paris Bettencourt/Human Practice/perception2012-10-26T22:08:28Z<p>Aleksandra: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="boston"><br />
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<br />
<br />
<br />
During this summer, all of us gained knowledge in synthetic biology and learned lab skills, but that wasn't all. <br />
From the beginning of our brainstorming sessions, safety questions came up in our discussions. Our mutual interest in this topic lead us to center our project on safeguard systems and human practices related to public awareness and risk assesssment. This meant that we had to work hard not only on our wet lab project, but also on human practices.<br />
The greatest thing is, this effort resulted not only in the outreach towards lay people, but also changed our own opinion on biosafety in the context of synthetic biology. We feel that our Human Practice project make a change for each and every one of us. Here are our personal perceptions.<br />
<br />
[[Image:Julianne.png|thumb|left|100px]] When we first began our project, I was really skeptical about the long term goals of releasing bacteria into the environment. However, during the debate we held some members of the "government", arguing in favor of the release of genetically modified bacteria, reminded the audience that there was a time when some held the opinion that airplanes were infernal machines that would only end in doom. We were questioned by judges in Amsterdam, who said that bacterial containment is impossible. I was inspired by the debaters. We have two options, we can either accept that biological containment is impossible, or we can try to study this problem and develop containment devices. In the end we may come to the conclusion that the risks are too great to ever release GE bacteria into the environment, but if we do not try to explore this problem we will do a great disservice to all the beautiful and brilliant iGEM projects dedicated to bioremediation. <br />
<br />
<br />
<br />
[[Image:Ernest.png|thumb|left|100px]] Human practices definitely brought a new dimension to our project. The question of biosafety is too broad to be tackled from the "narrow" point of view of a pure synthetic biologist, and we realized how important the contact and the discussion with the population is when dealing with such a sensitive topic. In our case, both the risks and the potential benefits are huge, leading to very polarized opinions among the interviewees. I strongly believe that scientists must make an effort to increase the transparency of their results and not push their ideas if the population doesn't accept them, in order to reduce the gap that now separates them from the rest of the population.<br />
<br />
<br />
[[Image:Aishah.png|thumb|left|100px]] The most remarkable thing I learned from our human practice project is the level of public awareness on genetic modification. I am not a biologist myself, and before I did not really care about this field--I always thought 'the experts knows better'. But after seeing the debate and even the discussion among high school students about this field, I was surprised with their opinion showing how much they actually aware. I guess it may be a bit related with the different education culture in France and in my country where the students are less encouraged to speak about their thought; but as a prospective scientist I learned that I should care more about public opinion, as well as expose myself with new knowledge and information.<br />
<br />
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<br />
<br />
[[Image:Dylan.png|thumb|left|100px]] Our project was initially based upon the idea of genetically modified bacteria that could be sprayed along with DDT. The bacteria would degrade the DDT after some time, hopefully having a less drastic environmental impact, as the DDT would soon disappear. We started to consider consider human practices as an important issue and realized that the DDT project could have other unaddressed dangers. What if the DDT degrading genes were transferred to other species? DDT degrading gene could be transferred to mosquitoes, and then our system would have done something far, far worse than the its potential for good. But I know that there must be worthy risks in terms of environmental applications of genetically modified bacteria. Through human practices I learned that most people would agree, through a proper weighing of benefit vs. risk, certain projects should be applied in the environment as long as we use proper genetic safeguards for safety.<br />
<br />
<br />
[[Image:Claire.png|thumb|left|100px]] I could not imagine how anyone in his/her right mind could be opposed to synthetic biology and its applications. I was convinced that if people were, it was because they did not really know much about the field, because they are ignorant. Therefore if we educated them, they would realize how GREAT SB is and would accept it.<br />
Now, thanks to the human practice project, I realized that this naïve vision of things is completely false and also very dangerous!<br />
First realization: People have the legitimate right to be opposed to synthetic biology. There is no link between ignorance of SB and rejection of its applications<br />
Second realization: Every citizen should have a say in what technologies they want or do not want. Experts should not be the ones making the final call!<br />
Third realization: Education is very important. The aim should be to give people all the necessary tools to understand what exactly is going on, and so that they can therefore discuss in the most illuminated way possible if they want or not the technology as part of their world (education’s aim should absolutely not be making people agree with us and accept synthetic biology! This vision is dangerous!!!)<br />
<br />
[[Image:Denis.png|thumb|left|100px]] I came from Physics and until last year I didn't know anything about synthetic biology and biodegradation. However, I was always interested in projects intended to save the world. Or, at least, how to deal with problems caused by humanity? Due to that, I always was concerned about the big amount of waste produced by peoples. After I learned that Synthetic biology develops methods to solve those problems, I came up with the idea to degrade insecticide using bacteria, but with a delay: first, to kill insects, and after some delay, to degrade insecticide to avoid side effects. At that time, I had no idea about gene transfer, and that scientists don't release any synthetic bacteria to the environment. For me it was really surprising! How we could benefit from such great ideas like iGEM projects without having any possibility to use bacteria outside the lab? A lot of question appeared. Is it possible to create a safe containment system? What is the risk? Would ordinary citizens be interested in such projects? Those questions gave rise to our iGEM project, and human practice in parallel with theoretical and laboratory work partially gave me an answer to it.<br />
<br />
<br />
[[Image:Jean.png|thumb|left|100px]] What I found good from the Human practice part, was the interview we had with specialist, which was very interesting, because we could have had different point of views, and in the same time some really good and rich discussions. Also the report was good for me to keep a trace of the historical events that drive us in our situation. The debate was a good idea and we couldn't have expected more from it.<br />
Concerning teaching to the high school student synthetic biology, it's very disturbing for me, because in one hand, biotech companies give tools to high schools to build transgenic crops, in order to make their reputation better and not in a total altruistic way. In the other hand, we suggest to teach synthetic biology to kids, and for me it's hard to know whether it's really to teach them how to be critical toward this technology, or in fact doing the same as biotech companies, because they're still young and most of them won't see limits, even if they are taught.<br />
I think that at least, it should be taught in university for biologist, which is not done so far, unless being in a synthetic biology curriculum.<br />
<br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
</div><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/perceptionTeam:Paris Bettencourt/Human Practice/perception2012-10-26T22:07:40Z<p>Aleksandra: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="boston"><br />
<!-- ########## Don't edit above ########## --><br />
<br />
<br />
<br />
During this summer, all of us gained knowledge in synthetic biology and learned lab skills, but that wasn't all. <br />
From the beginning of our brainstorming sessions, safety questions came up in our discussions. Our mutual interest in this topic lead us to center our project on safeguard systems and human practices related to public awareness and risk assesssment. This meant that we had to work hard not only on our wet lab project, but also on human practices.<br />
The greatest thing is, this effort resulted not only in the outreach towards lay people, but also changed our own opinion on biosafety in the context of synthetic biology. We feel that our Human Practice project make a change for each and every one of us. Here are our personal perceptions.<br />
<br />
[[Image:Julianne.png|thumb|left|100px]] When we first began our project, I was really skeptical about the long term goals of releasing bacteria into the environment. However, during the debate we held some members of the "government", arguing in favor of the release of genetically modified bacteria, reminded the audience that there was a time when some held the opinion that airplanes were infernal machines that would only end in doom. We were questioned by judges in Amsterdam, who said that bacterial containment is impossible. I was inspired by the debaters. We have two options, we can either accept that biological containment is impossible, or we can try to study this problem and develop containment devices. In the end we may come to the conclusion that the risks are too great to ever release GE bacteria into the environment, but if we do not try to explore this problem we will do a great disservice to all the beautiful and brilliant iGEM projects dedicated to bioremediation. <br />
<br />
<br />
<br />
[[Image:Ernest.png|thumb|left|100px]] Human practices definitely brought a new dimension to our project. The question of biosafety is too broad to be tackled from the "narrow" point of view of a pure synthetic biologist, and we realized how important the contact and the discussion with the population is when dealing with such a sensitive topic. In our case, both the risks and the potential benefits are huge, leading to very polarized opinions among the interviewees. I strongly believe that scientists must make an effort to increase the transparency of their results and not push their ideas if the population doesn't accept them, in order to reduce the gap that now separates them from the rest of the population.<br />
<br />
<br />
[[Image:Aishah.png|thumb|left|100px]] The most remarkable thing I learned from our human practice project is the level of public awareness on genetic modification. I am not a biologist myself, and before I did not really care about this field--I always thought 'the experts knows better'. But after seeing the debate and even the discussion among high school students about this field, I was surprised with their opinion showing how much they actually aware. I guess it may be a bit related with the different education culture in France and in my country where the students are less encouraged to speak about their thought; but as a prospective scientist I learned that I should care more about public opinion, as well as expose myself with new knowledge and information.<br />
<br />
<br />
<br />
<br />
[[Image:Dylan.png|thumb|left|100px]] Our project was initially based upon the idea of genetically modified bacteria that could be sprayed along with DDT. The bacteria would degrade the DDT after some time, hopefully having a less drastic environmental impact, as the DDT would soon disappear. We started to consider consider human practices as an important issue and realized that the DDT project could have other unaddressed dangers. What if the DDT degrading genes were transferred to other species? DDT degrading gene could be transferred to mosquitoes, and then our system would have done something far, far worse than the its potential for good. But I know that there must be worthy risks in terms of environmental applications of genetically modified bacteria. Through human practices I learned that most people would agree, through a proper weighing of benefit vs. risk, certain projects should be applied in the environment as long as we use proper genetic safeguards for safety.<br />
<br />
<br />
<br />
<br />
[[Image:Claire.png|thumb|left|100px]] I could not imagine how anyone in his/her right mind could be opposed to synthetic biology and its applications. I was convinced that if people were, it was because they did not really know much about the field, because they are ignorant. Therefore if we educated them, they would realize how GREAT SB is and would accept it.<br />
Now, thanks to the human practice project, I realized that this naïve vision of things is completely false and also very dangerous!<br />
First realization: People have the legitimate right to be opposed to synthetic biology. There is no link between ignorance of SB and rejection of its applications<br />
Second realization: Every citizen should have a say in what technologies they want or do not want. Experts should not be the ones making the final call!<br />
Third realization: Education is very important. The aim should be to give people all the necessary tools to understand what exactly is going on, and so that they can therefore discuss in the most illuminated way possible if they want or not the technology as part of their world (education’s aim should absolutely not be making people agree with us and accept synthetic biology! This vision is dangerous!!!)<br />
<br />
<br />
[[Image:Denis.png|thumb|left|100px]] I came from Physics and until last year I didn't know anything about synthetic biology and biodegradation. However, I was always interested in projects intended to save the world. Or, at least, how to deal with problems caused by humanity? Due to that, I always was concerned about the big amount of waste produced by peoples. After I learned that Synthetic biology develops methods to solve those problems, I came up with the idea to degrade insecticide using bacteria, but with a delay: first, to kill insects, and after some delay, to degrade insecticide to avoid side effects. At that time, I had no idea about gene transfer, and that scientists don't release any synthetic bacteria to the environment. For me it was really surprising! How we could benefit from such great ideas like iGEM projects without having any possibility to use bacteria outside the lab? A lot of question appeared. Is it possible to create a safe containment system? What is the risk? Would ordinary citizens be interested in such projects? Those questions gave rise to our iGEM project, and human practice in parallel with theoretical and laboratory work partially gave me an answer to it.<br />
<br />
<br />
<br />
[[Image:Jean.png|thumb|left|100px]] What I found good from the Human practice part, was the interview we had with specialist, which was very interesting, because we could have had different point of views, and in the same time some really good and rich discussions. Also the report was good for me to keep a trace of the historical events that drive us in our situation. The debate was a good idea and we couldn't have expected more from it.<br />
Concerning teaching to the high school student synthetic biology, it's very disturbing for me, because in one hand, biotech companies give tools to high schools to build transgenic crops, in order to make their reputation better and not in a total altruistic way. In the other hand, we suggest to teach synthetic biology to kids, and for me it's hard to know whether it's really to teach them how to be critical toward this technology, or in fact doing the same as biotech companies, because they're still young and most of them won't see limits, even if they are taught.<br />
I think that at least, it should be taught in university for biologist, which is not done so far, unless being in a synthetic biology curriculum.<br />
<br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
</div><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/perceptionTeam:Paris Bettencourt/Human Practice/perception2012-10-26T22:06:04Z<p>Aleksandra: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="boston"><br />
<!-- ########## Don't edit above ########## --><br />
<br />
<br />
<br />
During this summer, all of us gained knowledge in synthetic biology and learned lab skills, but that wasn't all. <br />
From the beginning of our brainstorming sessions, safety questions came up in our discussions. Our mutual interest in this topic lead us to center our project on safeguard systems and human practices related to public awareness and risk assesssment. This meant that we had to work hard not only on our wet lab project, but also on human practices.<br />
The greatest thing is, this effort resulted not only in the outreach towards lay people, but also changed our own opinion on biosafety in the context of synthetic biology. We feel that our Human Practice project make a change for each and every one of us. Here are our personal perceptions.<br />
<br />
[[Image:Julianne.png|thumb|left|100px]] When we first began our project, I was really skeptical about the long term goals of releasing bacteria into the environment. However, during the debate we held some members of the "government", arguing in favor of the release of genetically modified bacteria, reminded the audience that there was a time when some held the opinion that airplanes were infernal machines that would only end in doom. We were questioned by judges in Amsterdam, who said that bacterial containment is impossible. I was inspired by the debaters. We have two options, we can either accept that biological containment is impossible, or we can try to study this problem and develop containment devices. In the end we may come to the conclusion that the risks are too great to ever release GE bacteria into the environment, but if we do not try to explore this problem we will do a great disservice to all the beautiful and brilliant iGEM projects dedicated to bioremediation. <br />
<br />
[[Image:Ernest.png|thumb|left|100px]] Human practices definitely brought a new dimension to our project. The question of biosafety is too broad to be tackled from the "narrow" point of view of a pure synthetic biologist, and we realized how important the contact and the discussion with the population is when dealing with such a sensitive topic. In our case, both the risks and the potential benefits are huge, leading to very polarized opinions among the interviewees. I strongly believe that scientists must make an effort to increase the transparency of their results and not push their ideas if the population doesn't accept them, in order to reduce the gap that now separates them from the rest of the population.<br />
<br />
[[Image:Aishah.png|thumb|left|100px]] The most remarkable thing I learned from our human practice project is the level of public awareness on genetic modification. I am not a biologist myself, and before I did not really care about this field--I always thought 'the experts knows better'. But after seeing the debate and even the discussion among high school students about this field, I was surprised with their opinion showing how much they actually aware. I guess it may be a bit related with the different education culture in France and in my country where the students are less encouraged to speak about their thought; but as a prospective scientist I learned that I should care more about public opinion, as well as expose myself with new knowledge and information.<br />
<br />
[[Image:Dylan.png|thumb|left|100px]] Our project was initially based upon the idea of genetically modified bacteria that could be sprayed along with DDT. The bacteria would degrade the DDT after some time, hopefully having a less drastic environmental impact, as the DDT would soon disappear. We started to consider consider human practices as an important issue and realized that the DDT project could have other unaddressed dangers. What if the DDT degrading genes were transferred to other species? DDT degrading gene could be transferred to mosquitoes, and then our system would have done something far, far worse than the its potential for good. But I know that there must be worthy risks in terms of environmental applications of genetically modified bacteria. Through human practices I learned that most people would agree, through a proper weighing of benefit vs. risk, certain projects should be applied in the environment as long as we use proper genetic safeguards for safety.<br />
<br />
[[Image:Claire.png|thumb|left|100px]] I could not imagine how anyone in his/her right mind could be opposed to synthetic biology and its applications. I was convinced that if people were, it was because they did not really know much about the field, because they are ignorant. Therefore if we educated them, they would realize how GREAT SB is and would accept it.<br />
Now, thanks to the human practice project, I realized that this naïve vision of things is completely false and also very dangerous!<br />
First realization: People have the legitimate right to be opposed to synthetic biology. There is no link between ignorance of SB and rejection of its applications<br />
Second realization: Every citizen should have a say in what technologies they want or do not want. Experts should not be the ones making the final call!<br />
Third realization: Education is very important. The aim should be to give people all the necessary tools to understand what exactly is going on, and so that they can therefore discuss in the most illuminated way possible if they want or not the technology as part of their world (education’s aim should absolutely not be making people agree with us and accept synthetic biology! This vision is dangerous!!!)<br />
<br />
[[Image:Denis.png|thumb|left|100px]] I came from Physics and until last year I didn't know anything about synthetic biology and biodegradation. However, I was always interested in projects intended to save the world. Or, at least, how to deal with problems caused by humanity? Due to that, I always was concerned about the big amount of waste produced by peoples. After I learned that Synthetic biology develops methods to solve those problems, I came up with the idea to degrade insecticide using bacteria, but with a delay: first, to kill insects, and after some delay, to degrade insecticide to avoid side effects. At that time, I had no idea about gene transfer, and that scientists don't release any synthetic bacteria to the environment. For me it was really surprising! How we could benefit from such great ideas like iGEM projects without having any possibility to use bacteria outside the lab? A lot of question appeared. Is it possible to create a safe containment system? What is the risk? Would ordinary citizens be interested in such projects? Those questions gave rise to our iGEM project, and human practice in parallel with theoretical and laboratory work partially gave me an answer to it.<br />
<br />
[[Image:Jean.png|thumb|left|100px]] What I found good from the Human practice part, was the interview we had with specialist, which was very interesting, because we could have had different point of views, and in the same time some really good and rich discussions. Also the report was good for me to keep a trace of the historical events that drive us in our situation. The debate was a good idea and we couldn't have expected more from it.<br />
Concerning teaching to the high school student synthetic biology, it's very disturbing for me, because in one hand, biotech companies give tools to high schools to build transgenic crops, in order to make their reputation better and not in a total altruistic way. In the other hand, we suggest to teach synthetic biology to kids, and for me it's hard to know whether it's really to teach them how to be critical toward this technology, or in fact doing the same as biotech companies, because they're still young and most of them won't see limits, even if they are taught.<br />
I think that at least, it should be taught in university for biologist, which is not done so far, unless being in a synthetic biology curriculum.<br />
<br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
</div><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/perceptionTeam:Paris Bettencourt/Human Practice/perception2012-10-26T21:56:21Z<p>Aleksandra: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="boston"><br />
<!-- ########## Don't edit above ########## --><br />
<br />
During this summer, all of us gained knowledge in synthetic biology and learned lab skills, but that wasn't all. <br />
From the beginning of our brainstorming sessions, safety questions came up in our discussions. Our mutual interest in this topic lead us to center our project on safeguard systems and human practices related to public awareness and risk assesssment. This meant that we had to work hard not only on our wet lab project, but also on human practices.<br />
The greatest thing is, this effort resulted not only in the outreach towards lay people, but also changed our own opinion on biosafety in the context of synthetic biology. We feel that our Human Practice project make a change for each and every one of us. Here are our personal perceptions.<br />
<br />
[[Image:Julianne.png|thumb|left|100px]] When we first began our project, I was really skeptical about the long term goals of releasing bacteria into the environment. However, during the debate we held some members of the "government", arguing in favor of the release of genetically modified bacteria, reminded the audience that there was a time when some held the opinion that airplanes were infernal machines that would only end in doom. We were questioned by judges in Amsterdam, who said that bacterial containment is impossible. I was inspired by the debaters. We have two options, we can either accept that biological containment is impossible, or we can try to study this problem and develop containment devices. In the end we may come to the conclusion that the risks are too great to ever release GE bacteria into the environment, but if we do not try to explore this problem we will do a great diservice to all the beautiful and brilliant iGEM projects dedicated to bioremediation. <br />
<br />
<!-- ########## Don't edit below ########## --><br />
</div><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T03:35:24Z<p>Aleksandra: /* Results */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
We designed a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid. In our specific containment module, this will result in extinguishing the expression of the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. This will lead to activation of the colicin DNAse toxin thst will degrade the cell's chromosomal DNA, leading to its death.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*10^10 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===I-SceI is expressed and active in eliminating restriction-site harbouring plasmid===<br />
To measure the digestion efficiency of I-SceI, we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was a kind gift of the [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team] which we further characterized.<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
First, we decided to check the hypothesis 1, and verify if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
We followed the following protocol:<br />
<br />
* Pick a colony and start a liquid culture with both antibiotic resistances (Amp and Cm).<br />
* Incubate at 37°C until optical density (OD) reaches 0.5<br />
* Pellet and wash cells to remove Amp and Cm.<br />
* Re-dilute them in the same volume of LB.<br />
* From each of those two tubes, start two liquid cultures:<br />
*# The first culture with Cm and w/o IPTG.<br />
*# The second culture with Cm and IPTG.<br />
* Incubate overnight at 37°C<br />
<br />
=====Step 3=====<br />
<br />
Plate colonies from each tube on two different plates:<br />
# Selection with Cm and Amp.<br />
# Selection with Cm.<br />
<br />
==== Results ====<br />
<br />
To analyze data, we counted and compared the number of colonies on four plates corresponding to 4 conditions.<br />
<br>First, we compared the number of CFU formed by non-induced cells, plated with a single antibiotic and with both antibiotics. We expected that the plate with only Cm selection would have the biggest number of colonies. A smaller number would be on the plate with two antibiotics (Amp and Cm). It could be explained by the loss of the plasmid carrying Amp resistance.<br />
<br>Next, we compared the number of CFUs formed by the cells where we induced the I-SceI expression, plated with a single antibiotic and with both antibiotics. We expect that there would be much less colonies on the double antibiotic plate, because the Ampicillin carrying plasmid would be lost not only due to the absence of selection during growth (as for the first two cells), but also due to the digestion by the restriction enzyme.<br />
<br />
<br />
=====The combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|Selection: Cm <br/> Plated colonies: w/o IPTG]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|Selection: Cm <br/> Plated colonies: IPTG induced]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|Selection: Cm & Amp <br/> Plated colonies: w/o IPTG]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp <br/> Plated colonies: IPTG induced]]<br />
|}<br />
<br />
From photos above we can see that our expectation came true.<br />
<br />
===Characterization of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
{|align="center"<br />
|-valign="top"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
[[Image:Paris_Bettencourt_2012_RG_pRha_graph_3.jpg|thumb|530px|center|<font size="1">Characterized the pRha promoter using a plate reader</font>]]<br />
<br />
As we can see from the graph above, pRha promoter works as expected and it could be well tuned by concentration of L-rhamnose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/AttributionsTeam:Paris Bettencourt/Attributions2012-09-27T03:19:14Z<p>Aleksandra: /* Collaborations */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="grouptitle">Attributions</div><br />
==Collaborations==<br />
The collaborations of our team this year was based on mutual exchange on containment, safety and human practices in general in the synthetic biology realm. Our lovely collaborators:<br />
* iGEM Grenoble team: <br />
*:We helped Jerome and Nadia improve their safety sheet proposal for the parts registry, considering the content, layout, and its accessibility. <br />
*:Team members came to our debate, and were part of the adjudication pannel, they gave us detailed feedback at the end.<br />
<br />
* iGEM UCL team: <br />
*:Bethan and Philipp came to Paris! We helped them set up meetings with researchers from [http://www.lapaillasse.org/ La Paillasse (DIY bio lab)] and [http://fabelier.org/ Fabelier (DIY tinkerers)] and offered them crash. <br />
*:Philipp kindly participated in the debate as part of the adjudication pannel, and Bethan brought an interesting perspective to the post-debate discussion. <br />
[[Image:UCLParisiGEM2012.jpg| 300 px |center]]<br />
<br />
<br />
* iGEM TUDelft team:<br />
*:They've kindly sent us two biobricks from the 2009 team: the [http://partsregistry.org/Part:BBa_K175041 I-SceI restriction enzyme] and the [http://partsregistry.org/Part:BBa_K175027 corresponding restriction site]. <br />
<html><center><a href="https://static.igem.org/mediawiki/igem.org/8/8e/Paris_Bettencourt.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/igem.org/8/8e/Paris_Bettencourt.jpg" align="center" width="50%"></a></center></html><br />
<br />
==Responsibilities in the Team==<br />
<br />
All of the designs, constructs (unless stated otherwise) and experiments presented in this wiki were performed by the members of the 2012 Paris Bettencourt team. The advisors and instructors were providing feedback and advice, when needed. None of the subjects of this project are being studied or developed in the hosting lab.<br />
<br />
All members of the team were following the development of the project as a whole. However, in order to be able to work on several modules simultaneously, we formed teams of 1 or 2 that were mainly responsible for each part. <br />
<br />
<div><b>Delay system:</b> Ernest Mordret<br />
<div><b>Semantic containment:</b> Jean Cury<br />
<div><b>Restriction enzyme system:</b> Denis Samuylov and Claire Mayer<br />
<div><b>MAGE:</b> Guillaume Villain and Zoran Marinkovic<br />
<div><b>Suicide system:</b> Julianne Rieders and Aishah Prastowo<br />
<div><b>Encapsulation:</b> Dylan Iverson<br />
<div><b>Synthetic Import Domain:</b> Zoran Marinkovic and Guillaume Villain<br />
<div><b>Human practice:</b> Claire Mayer and Jean Cury<br />
<div><b>Wiki layout:</b> Jean Cury<br />
<div><b>Stop motion:</b> Dylan Iverson, Jean Cury and Julianne Rieders<br />
<div><b>Bonus:</b> Aishah Prastowo and Julianne Rieders<br />
<br />
==External help==<br />
We are extremely thankful to all the following labs, iGEM teams, researchers we met, and other people, for their help :<br />
* Sara Aguiton for her precious advice on the human practice report.<br />
* Professor Mamzer-Bruneel, Professor Gouyon, Professor Morange, Professor Ricroch for the interviews.<br />
* Professor Yokobayashi, for the sRNA repression plasmidic system<br />
* Osnat Gillor, for the Colicin E2 strains<br />
* Miklos de Zamaroczy, for the Colicin D strain<br />
* Bethan and Philip from the UCL iGEM team for participating in our debate and for their discusion and feedback.<br />
* Grégory Hansen and Jean-Baptise Lugagne from the grenoble iGEM team for participating in our debate, and for their feedback.<br />
* Dr. Rosenberg, for the SMR6316 strain with encoded I-SceI endonuclease.<br />
* Dr. Josef Altenbuchner, for the plasmid pJOE3075 with encoded Rhamnose promoter.<br />
* Esengul Yildirim and TUDelft iGEM 2012 team for sending us two biobricks: [http://partsregistry.org/Part:BBa_K175027 BBa_K175027] and [http://partsregistry.org/Part:BBa_K175041 BBa_K175041].<br />
* Theo Sanderson of Cambridge iGEM 2010 team for advice on use of the lux brick.<br />
* Myelin Haoqian Zhang for help with mercury project.<br />
* Bristol 2010 iGEM team for their wonderful Nitrate reporter.<br />
<!--Each team must clearly attribute work done by the team on this page. They must distinguish work done by the team from work done by others, including the host labs, advisors, instructors, graduate students, and postgraduate masters students.--><br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/AttributionsTeam:Paris Bettencourt/Attributions2012-09-27T03:14:16Z<p>Aleksandra: /* Collaborations */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="grouptitle">Attributions</div><br />
==Collaborations==<br />
The collaborations of our team this year was based on mutual exchange on containment, safety and human practices in general in the synthetic biology realm. Our lovely collaborators:<br />
* iGEM Grenoble team: <br />
*:We helped Jerome and Nadia improve their safety sheet proposal for the parts registry, considering the content, layout, and its accessibility. <br />
*:Team members came to our debate, and were part of the adjudication pannel, they gave us detailed feedback at the end.<br />
<br />
* iGEM UCL team: <br />
*:Bethan and Philipp came to Paris! We helped them set up meetings with researchers from [http://www.lapaillasse.org/ La Paillasse (DIY bio lab)] and [http://fabelier.org/ Fabelier (DIY tinkerers)] and offered them crash. <br />
*:Philipp kindly participated in the debate as part of the adjudication pannel, and Bethan brought an interesting perspective to the post-debate discussion. <br />
[[Image:UCLParisiGEM2012.jpg| 300 px |center]]<br />
<br />
<br />
* iGEM TUDelft team:<br />
*:They've kindly sent us two biobricks from the 2009 team: the I-SceI restriction enzyme and the corresponding restriction site. <br />
<html><center><a href="https://static.igem.org/mediawiki/igem.org/8/8e/Paris_Bettencourt.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/igem.org/8/8e/Paris_Bettencourt.jpg" align="center" width="50%"></a></center></html><br />
<br />
==Responsibilities in the Team==<br />
<br />
All of the designs, constructs (unless stated otherwise) and experiments presented in this wiki were performed by the members of the 2012 Paris Bettencourt team. The advisors and instructors were providing feedback and advice, when needed. None of the subjects of this project are being studied or developed in the hosting lab.<br />
<br />
All members of the team were following the development of the project as a whole. However, in order to be able to work on several modules simultaneously, we formed teams of 1 or 2 that were mainly responsible for each part. <br />
<br />
<div><b>Delay system:</b> Ernest Mordret<br />
<div><b>Semantic containment:</b> Jean Cury<br />
<div><b>Restriction enzyme system:</b> Denis Samuylov and Claire Mayer<br />
<div><b>MAGE:</b> Guillaume Villain and Zoran Marinkovic<br />
<div><b>Suicide system:</b> Julianne Rieders and Aishah Prastowo<br />
<div><b>Encapsulation:</b> Dylan Iverson<br />
<div><b>Synthetic Import Domain:</b> Zoran Marinkovic and Guillaume Villain<br />
<div><b>Human practice:</b> Claire Mayer and Jean Cury<br />
<div><b>Wiki layout:</b> Jean Cury<br />
<div><b>Stop motion:</b> Dylan Iverson, Jean Cury and Julianne Rieders<br />
<div><b>Bonus:</b> Aishah Prastowo and Julianne Rieders<br />
<br />
==External help==<br />
We are extremely thankful to all the following labs, iGEM teams, researchers we met, and other people, for their help :<br />
* Sara Aguiton for her precious advice on the human practice report.<br />
* Professor Mamzer-Bruneel, Professor Gouyon, Professor Morange, Professor Ricroch for the interviews.<br />
* Professor Yokobayashi, for the sRNA repression plasmidic system<br />
* Osnat Gillor, for the Colicin E2 strains<br />
* Miklos de Zamaroczy, for the Colicin D strain<br />
* Bethan and Philip from the UCL iGEM team for participating in our debate and for their discusion and feedback.<br />
* Grégory Hansen and Jean-Baptise Lugagne from the grenoble iGEM team for participating in our debate, and for their feedback.<br />
* Dr. Rosenberg, for the SMR6316 strain with encoded I-SceI endonuclease.<br />
* Dr. Josef Altenbuchner, for the plasmid pJOE3075 with encoded Rhamnose promoter.<br />
* Esengul Yildirim and TUDelft iGEM 2012 team for sending us two biobricks: [http://partsregistry.org/Part:BBa_K175027 BBa_K175027] and [http://partsregistry.org/Part:BBa_K175041 BBa_K175041].<br />
* Theo Sanderson of Cambridge iGEM 2010 team for advice on use of the lux brick.<br />
* Myelin Haoqian Zhang for help with mercury project.<br />
* Bristol 2010 iGEM team for their wonderful Nitrate reporter.<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/OverviewTeam:Paris Bettencourt/Overview2012-09-27T03:01:24Z<p>Aleksandra: /* Key Killswitch Design Features */</p>
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1b/MOUSECURSORPB12.png" width="60px" border="0" /><span></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/e/e3/PhysicalContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/f/f6/PhysicalContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV"><b>1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth</b> </div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/0/09/SemanticContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br><b>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. </b></div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
<b>3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.</b><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/5/5c/RestrictionSystem.png" width="60px" border="0" /><span><img src="/wiki/images/9/97/Restriction2PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
<b>4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.</b></div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/d/da/SkullIcon.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.<br><br><b>5) Suicide system: Once the anti-toxin concentration is below a given threshold, the toxin is no longer inhibited. It kills the cell as well as its neighbors, and eliminates extracellular DNA via its DNase activity.</b></div></span></a><br />
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==Our project - a hypothetical case study==<br />
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Imagine a farmer that wants measure the nutrient concentration in her field, the better to optimize her fertilizer use. We could provide her with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in alginate beads. She would spread the beads in her field, wait for 12 hours, and then check if they are glowing in response to the nitrates in the soil.<br />
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We want this system to work the way the original designers intended. We also want to reduce the chance that the engineered bacteria will survive in the soil, release intact DNA, or transfer genes to a soil microbe.<br />
<br />
Our system will be implemented as a pair of plasmids, compatible with the most commonly used BioBrick plasmids, and therefore easily integrated with existing systems. The AgrEColi are now running bWARE, and express a new containment functionality. <br />
<br />
Shortly after the beads enter the soid, the delay system triggers and the DNA-degrading colicin proteins become active. The colicins specifically recognize and enter E. coli cells, killing them by completely degrading their genomes. The colicins also degrade DNA loose in the environment, without harming the native bacterial species.<br />
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Every field is a different ecosystem with a different composition of native species. The potential consequences of horizontal gene transfer are therefore difficult to predict in general. By destroying the information contained in DNA, our system reduces the chance that introduced DNA will replicate in the new environment.<br />
<br />
==Objectives==<br />
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Our project aims to:<br />
<br />
*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
<br />
To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
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====Key Killswitch Design Features====<br />
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*The DNAse toxin kills cells and destroys genetic information.<br />
*A population-level mechanism compensates system failure in single cells.<br />
*Plasmid destruction with restriction enzymes is an irreversible trigger.<br />
*A specific toxin targeting mechanism reduces system impact on native fauna.<br />
*The modular design supports integration with existing iGEM projects.<br />
*The use of several redundant modules compensates the failure of one of them.<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T02:52:34Z<p>Aleksandra: /* Step 3 */</p>
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<div id="grouptitle">Restriction Enzyme System</div><br />
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<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
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* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
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==Overview==<br />
We designed a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid. In our specific containment module, this will result in extinguishing the expression of the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. This will lead to activation of the colicin DNAse toxin thst will degrade the cell's chromosomal DNA, leading to its death.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*10^10 base pairs or once in 20 human genomes.</li><br />
</ol><br />
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<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
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<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
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<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
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</li><br />
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<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
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<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
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==Experiments and results==<br />
<br />
===I-SceI is expressed and active in eliminating restriction-site harbouring plasmid===<br />
To measure the digestion efficiency of I-SceI, we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was a kind gift of the [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team] which we further characterized.<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
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{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
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=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
First, we decided to check the hypothesis 1, and verify if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
We followed the following protocol:<br />
<br />
* Pick a colony and start a liquid culture with both antibiotic resistances (Amp and Cm).<br />
* Incubate at 37°C until optical density (OD) reaches 0.5<br />
* Pellet and wash cells to remove Amp and Cm.<br />
* Re-dilute them in the same volume of LB.<br />
* From each of those two tubes, start two liquid cultures:<br />
*# The first culture with Cm and w/o IPTG.<br />
*# The second culture with Cm and IPTG.<br />
* Incubate overnight at 37°C<br />
<br />
=====Step 3=====<br />
<br />
Plate colonies from each tube on two different plates:<br />
# Selection with Cm and Amp.<br />
# Selection with Cm.<br />
<br />
==== Results ====<br />
<br />
To analyze data we counted and compared number of colonies on four plates. We expected that on plate with Cm selection with culture plated from the tube w/o IPTG we will get the biggest number of colonies.<br />
<br />
=====The combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|Selection: Cm <br/> Plated colonies: w/o IPTG]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|Selection: Cm <br/> Plated colonies: IPTG induced]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|Selection: Cm & Amp <br/> Plated colonies: w/o IPTG]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp <br/> Plated colonies: IPTG induced]]<br />
|}<br />
<br />
===Characterization of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
{|align="center"<br />
|-valign="top"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
[[Image:Paris_Bettencourt_2012_RG_pRha_graph_3.jpg|thumb|530px|center|<font size="1">Characterized the pRha promoter using a plate reader</font>]]<br />
<br />
As we can see from the graph above, pRha promoter works as expected and it could be well tuned by concentration of L-rhamnose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T02:52:00Z<p>Aleksandra: /* Step 2 */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
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<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
We designed a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid. In our specific containment module, this will result in extinguishing the expression of the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. This will lead to activation of the colicin DNAse toxin thst will degrade the cell's chromosomal DNA, leading to its death.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*10^10 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===I-SceI is expressed and active in eliminating restriction-site harbouring plasmid===<br />
To measure the digestion efficiency of I-SceI, we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was a kind gift of the [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team] which we further characterized.<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
First, we decided to check the hypothesis 1, and verify if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
We followed the following protocol:<br />
<br />
* Pick a colony and start a liquid culture with both antibiotic resistances (Amp and Cm).<br />
* Incubate at 37°C until optical density (OD) reaches 0.5<br />
* Pellet and wash cells to remove Amp and Cm.<br />
* Re-dilute them in the same volume of LB.<br />
* From each of those two tubes, start two liquid cultures:<br />
*# The first culture with Cm and w/o IPTG.<br />
*# The second culture with Cm and IPTG.<br />
* Incubate overnight at 37°C<br />
<br />
=====Step 3=====<br />
<br />
Plates colonies from each tube on two different plates:<br />
# Selection with Cm and Amp.<br />
# Selection with Cm.<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
To analyze data we counted and compared number of colonies on four plates. We expected that on plate with Cm selection with culture plated from the tube w/o IPTG we will get the biggest number of colonies.<br />
<br />
=====The combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|Selection: Cm <br/> Plated colonies: w/o IPTG]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|Selection: Cm <br/> Plated colonies: IPTG induced]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|Selection: Cm & Amp <br/> Plated colonies: w/o IPTG]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp <br/> Plated colonies: IPTG induced]]<br />
|}<br />
<br />
===Characterization of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
{|align="center"<br />
|-valign="top"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
[[Image:Paris_Bettencourt_2012_RG_pRha_graph_3.jpg|thumb|530px|center|<font size="1">Characterized the pRha promoter using a plate reader</font>]]<br />
<br />
As we can see from the graph above, pRha promoter works as expected and it could be well tuned by concentration of L-rhamnose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T02:50:09Z<p>Aleksandra: /* Step 2 */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
We designed a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid. In our specific containment module, this will result in extinguishing the expression of the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. This will lead to activation of the colicin DNAse toxin thst will degrade the cell's chromosomal DNA, leading to its death.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*10^10 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===I-SceI is expressed and active in eliminating restriction-site harbouring plasmid===<br />
To measure the digestion efficiency of I-SceI, we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was a kind gift of the [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team] which we further characterized.<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
First, we decided to check the hypothesis 1, and verify if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
We followed the following protocol:<br />
<br />
* Pick a colony and start a liquid culture with both antibiotic resistances (Amp and Cm).<br />
* Wait until optical density (OD) reaches 0.5<br />
* Pellet and wash cells to remove Amp and Cm.<br />
* Re-dilute them in the same volume of LB.<br />
* From each of those two tubes, start two liquid cultures:<br />
*# The first culture with Cm and w/o IPTG.<br />
*# The second culture with Cm and IPTG.<br />
* Wait overnight and plate<br />
<br />
=====Step 3=====<br />
<br />
Plates colonies from each tube on two different plates:<br />
# Selection with Cm and Amp.<br />
# Selection with Cm.<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
To analyze data we counted and compared number of colonies on four plates. We expected that on plate with Cm selection with culture plated from the tube w/o IPTG we will get the biggest number of colonies.<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
===Characterization of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
{|align="center"<br />
|-valign="top"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
[[Image:Paris_Bettencourt_2012_RG_pRha_graph_3.jpg|thumb|530px|center|<font size="1">Characterized the pRha promoter using a plate reader</font>]]<br />
<br />
As we can see from the graph above, pRha promoter works as expected and it could be well tuned by concentration of L-rhamnose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T02:47:17Z<p>Aleksandra: /* The third combination of plasmids: */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
We designed a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid. In our specific containment module, this will result in extinguishing the expression of the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. This will lead to activation of the colicin DNAse toxin thst will degrade the cell's chromosomal DNA, leading to its death.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*10^10 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===I-SceI is expressed and active in eliminating restriction-site harbouring plasmid===<br />
To measure the digestion efficiency of I-SceI, we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was a kind gift of the [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team] which we further characterized.<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
First, we decided to check the hypothesis 1, and verify if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
<br />
* Picke a colony and start a liquid culture with both antibiotic resistance Amp and Cm.<br />
* Waite until optical density (OD) reaches 0.5.<br />
* Wash cells to remove Amp and Cm.<br />
* Put back the same volume of LB.<br />
* From this tube start two liquid culture:<br />
*# The first culture with Cm and w/o IPTG.<br />
*# The second culture with Cm and IPTG.<br />
* Wait overnight<br />
<br />
=====Step 3=====<br />
<br />
Plates colonies from each tube on two different plates:<br />
# Selection with Cm and Amp.<br />
# Selection with Cm.<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
To analyze data we counted and compared number of colonies on four plates. We expected that on plate with Cm selection with culture plated from the tube w/o IPTG we will get the biggest number of colonies.<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
===Characterization of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
{|align="center"<br />
|-valign="top"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
[[Image:Paris_Bettencourt_2012_RG_pRha_graph_3.jpg|thumb|530px|center|<font size="1">Characterized the pRha promoter using a plate reader</font>]]<br />
<br />
As we can see from the graph above, pRha promoter works as expected and it could be well tuned by concentration of L-rhamnose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/AchievementsTeam:Paris Bettencourt/Achievements2012-09-27T02:40:25Z<p>Aleksandra: /* Achievements of all the different modules */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<br />
<div id="grouptitle">Achievements</div><br />
<br />
==Achievements of all the different modules==<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Semantic containment'''<br />
<br />
'''Aims :'''<br />
<br />
Creating a semantic containment system to prevent gene expression in natural organisms<br />
Characterize the system<br />
Use this system in all genes of the system, the critical genes first (e.g. colicin)<br />
System<br />
<br />
An amber codon (stop codon) embedded in protein genes to prevent their expression and an amber suppressor system in our genetically engineered bacteria<br />
<br />
'''Achievements :'''<br />
<br />
Construction and characterization of 2 biobricks :<br />
K914000 : PLac-supD-T : tRNA amber suppressor<br />
K914009 : P1003* Ser133->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
Both part were well characterized and works well. For the second parts, we show that as expected, one mutation is quite leaky, although it works qualitatively, but one mutation is not enough if we want to release such parts in nature. Other reasons emphasize this observation, notably the weakness of being at one mutation to recover the protein functionality.<br />
<br />
Creation of a new category in the part registry : Semantic containment. The aim of this category is to let people improving each part by adding for instance other amber mutations to existing part to increase the containment.<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Suicide system'''<br />
<br />
'''Aims :'''<br />
Implement a kill-switch that features population-level suicide and complete genome degradation. <br />
<br />
'''System :'''<br />
A synthetic toxin-anti-toxin system based on the wild type Colicin E2 operon.<br />
<br />
'''Achievements :'''<br />
We showed that Colicin E2 cells induce cell death in sensitive populations, and that these sensitive populations can be protected by providing them with our engineered immunity protein. <br />
* Construction of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914001 K914001] : pLac-repressilator RBS-Colicin E2 immunity protein<br />
** [http://partsregistry.org/Part:BBa_K914002 K914002] :repressilator RBS-Colicin E2 immunity protein<br />
Part K914001 is well characterized and provides immunity to sensitive cells against the Colicin E2 activity protein, but is leaky. Part K914002 is promoterless and allows users to easily plug in the appropriate promoter for their desired purpose. <br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety XNase]. The aim of this category is to provide users with DNase/RNase parts that can be used for improved kill switches featuring the degradation of genomic material.<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Restriction Enzyme System </b> <br />
<br />
<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
</tr><br />
</table><br />
<br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
'''MAGE'''<br />
<br />
'''Aims :'''<br />
<br />
Removal of four FseI restriction sites from E. coli MG1655 genome.<br />
<br />
'''Experimental System:'''<br />
<br />
Using multiplex automated genome engineering (MAGE) - a technique capable of editing the genome by making small changes in existing genomic sequences.<br />
<br />
'''Achievements:'''<br />
<br />
Proof of concept by introducing a stop codon in the middle of the lacZ gene<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
'''Synthetic Import Domain'''<br />
'''Aim :'''<br />
<br />
Creation of a novel protein import mechanism in bacteria. <br />
<br />
<br />
'''Experimental System:'''<br />
<br />
Exploit the natural Colicin import domain fused to any protein at will, dubbed here: "Synthetic Import Domain".<br />
<br />
'''Achievements:'''<br />
<br />
*Construction of colicin-like toxin by fusing Colicin E2 based "Synthetic Import Domain" with RNAse domain of colicin D<br />
*Constructon of FseI, I-SceI, LuxR active fragment, LacZ alpha fragment, PyrF and T7 RNA polymerase fused to the two types of "Synthetic Import Domains" from Colicin E2 and Colicin D<br />
*Proof of concept with LacZ alpha fragment fused to "Synthetic Import Domain" from Colicin D<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Encapsulation'''<br />
'''Aim:'''<br />
Harness bacteria-containing gel beads to assure cell containment and complement activity of genetic safety systems.<br />
<br />
'''Experimental system:'''<br />
Bacterial cells are encapsulated in alginate beads. We used a [https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation#Cell_Containment_Assay cell containment assay] based on plating to assess the release of cells from alginate beads. In addition, we aimed at improving the entrapment of cells through stabilization by polyethyleneimine and covalent cross-linkage by glutaraldehyde. <br />
<br />
'''Achievements:'''<br />
*Encapsulated cells achieved and their ability to propagate and express proteins within alginate beads demonstrated.<br />
*Stabilized alginate beads by covalent cross-linkage achieved and their ability to entrap cells demonstrated.<br />
*we performed [https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation#Bristol_2010_Nitrate_Reporter additional characterization] of the Bristol 2010 nitrate reporter [http://partsregistry.org/Part:BBa_K381001 K381001]<br />
* Efficient killing by colicin producing cells was achieved within the beads.<br />
</td><br />
</tr><br />
</table><br />
<br />
==Human Practice==<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b>Aim</b><br />
<br />
To chart new venues of best practice for synthetic biology. To this end, we examined the ethical, biological and social concerns related to the release of genetically modified bacteria in the wild.<br />
<br />
<b>Metodology</b><br />
<br />
#'''''Interviews with experts''''' which enabled us to have a broad overview of the state of the art. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Interview Read More]<br />
#'''''Interaction with high-schoolers''''' to have first-hand appreciation of reactions from first exposure to synthetic biology<br />
#'''''We screened previous iGEM team’s wikis''''' to trace the evolution of biosafety concerns and devices in the iGEM community, focusing on proposed containment systems. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/WikiScreen Read More]<br />
#'''''We focused on horizontal gene transfer as main generic risk factor'''''. <br />
#'''''Synthetic report''''' where we addressed the concerns raised by synthetic biology per se, that is, as a technique. Then, we analyzed the specific concerns that arise from synthetic biology’s potential applications in nature. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report Read More]<br />
<br />
<br />
<b>Main Conclusions</b><br />
# Societal interaction: <br />
#:*'''''The need to raise awareness''''' of synthetic biology in the population so people can decide in the most enlightened way possible if they want of this new technology and of its applications (A),<br />
#:* '''''The need of a discussion''''' between society’s different protagonists to set goals, define what they would consider as benefits and acceptable risks (B),<br />
# Best research practice:<br />
#:* '''''Zero risk is impossible to achieve''''' as no containment system can be 100% safe (bacteria can always escape by mutations) (C), <br />
#:* There is a '''''lack of quantitative data evaluating the probability of failure of any synthetic biology engineered system, in particular containment systems''''' (D),<br />
#:* There is a '''''lack of quantitative data evaluating the risk of HGT assuming containment systems failed''''' (E),<br />
#:* The compiling of the wiki screen shows that '''''no containment systems created in iGEM is robust''''': they lack the above quantification and are mostly one mutation away from failure. We call for major effort of the iGEM community to quantify available containment systems and search for new solutions (F),<br />
#:* '''''The need for an INDEPENDENT cohort of scientists''''' to test experimentally any application of synthetic biology that requires releasing in the environment (G), <br />
You can find the full list of conclusions [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report here]<br />
<br />
<b>Main Proposals</b><br />
# Societal interaction: <br />
#:* '''''Organizing a workshop''''' on synthetic biology and a tour of our lab for 60 high school students, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Workshop Read More]. First initiative for teaching synthetic biology in French high-school leading to a high-school iGEM team. Ultimately, we would like interaction with high school or middle school students to be a requirement for an iGEM gold medal. <br />
#:* '''''Organizing a debate''''' with 10 non expert students from various background, and then opening the debate to the floor (the public), which was made up of both experts and non experts, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Debate Read More]. <br />
#:* '''''Creating a page to explain horizontal gene transfer''''' to non scientists. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/HGT Go to HGT page]<br />
# Best research practice:<br />
#:* '''''Creating a system as robust as possible''''', that is many mutations away from failure (this is what our [https://2012.igem.org/Team:Paris_Bettencourt/Overview bench work] has been all about) (addresses issue C and F),<br />
#:* '''''Creating a safety page on the biobrick registry''''' where all the safety devices that exist are listed and characterized (included evaluation of their robustness) in order for iGEM teams to pick the most appropriate device to add to their newly created genetic circuit. Ultimately, we would like '''''the integration of safety modules and risks assessments to be part of of every synthetic biology project from the very start''''' (already listed in the safety page or created de novo by the team) (addresses issue D, F), [http://partsregistry.org/Biosafety Go to safety page]<br />
#:*The community has to '''''build a collection of bio-safety devices for future engineers'''''<br />
#:*Each synthetic biology application should '''''assess and disclose a list of application-specific risks and hazards'''''.<br />
#:*Development and adoption of a '''''safety chasis for synthetic biology research and prototyping'''''.<br />
<br />
You can find the full list of proposals [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report#III_Proposals here]<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/AchievementsTeam:Paris Bettencourt/Achievements2012-09-27T02:36:48Z<p>Aleksandra: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<br />
<div id="grouptitle">Achievements</div><br />
<br />
==Achievements of all the different modules==<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Semantic containment'''<br />
<br />
'''Aims :'''<br />
<br />
Creating a semantic containment system to prevent gene expression in natural organisms<br />
Characterize the system<br />
Use this system in all genes of the system, the critical genes first (e.g. colicin)<br />
System<br />
<br />
An amber codon (stop codon) embedded in protein genes to prevent their expression and an amber suppressor system in our genetically engineered bacteria<br />
<br />
'''Achievements :'''<br />
<br />
Construction and characterization of 2 biobricks :<br />
K914000 : PLac-supD-T : tRNA amber suppressor<br />
K914009 : P1003* Ser133->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
Both part were well characterized and works well. For the second parts, we show that as expected, one mutation is quite leaky, although it works qualitatively, but one mutation is not enough if we want to release such parts in nature. Other reasons emphasize this observation, notably the weakness of being at one mutation to recover the protein functionality.<br />
<br />
Creation of a new category in the part registry : Semantic containment. The aim of this category is to let people improving each part by adding for instance other amber mutations to existing part to increase the containment.<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Suicide system'''<br />
<br />
'''Aims :'''<br />
Implement a kill-switch that features population-level suicide and complete genome degradation. <br />
<br />
'''System :'''<br />
A synthetic toxin-anti-toxin system based on the wild type Colicin E2 operon.<br />
<br />
'''Achievements :'''<br />
We showed that Colicin E2 cells induce cell death in sensitive populations, and that these sensitive populations can be protected by providing them with our engineered immunity protein. <br />
* Construction of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914001 K914001] : pLac-repressilator RBS-Colicin E2 immunity protein<br />
** [http://partsregistry.org/Part:BBa_K914002 K914002] :repressilator RBS-Colicin E2 immunity protein<br />
Part K914001 is well characterized and provides immunity to sensitive cells against the Colicin E2 activity protein, but is leaky. Part K914002 is promoterless and allows users to easily plug in the appropriate promoter for their desired purpose. <br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety XNase]. The aim of this category is to provide users with DNase/RNase parts that can be used for improved kill switches featuring the degradation of genomic material.<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Restriction Enzyme System </b> <br />
<br />
<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
</tr><br />
</table><br />
<br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
'''MAGE'''<br />
<br />
'''Aims :'''<br />
<br />
Removal of four FseI restriction sites from E. coli MG1655 genome.<br />
<br />
'''Experimental System'''<br />
<br />
Using multiplex automated genome engineering (MAGE) - a technique capable of editing the genome by making small changes in existing genomic sequences.<br />
<br />
'''Achievements'''<br />
<br />
Proof of concept by introducing a stop codon in the middle of the lacZ gene<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
'''Synthetic Import Domain'''<br />
'''Aim :'''<br />
<br />
Creation of a novel protein import mechanism in bacteria. <br />
<br />
<br />
'''Experimental System'''<br />
<br />
Exploit the natural Colicin import domain fused to any protein at will, dubbed here: "Synthetic Import Domain".<br />
<br />
'''Achievements'''<br />
<br />
*Construction of colicin-like toxin by fusing Colicin E2 based "Synthetic Import Domain" with RNAse domain of colicin D<br />
*Constructon of FseI, I-SceI, LuxR active fragment, LacZ alpha fragment, PyrF and T7 RNA polymerase fused to the two types of "Synthetic Import Domains" from Colicin E2 and Colicin D<br />
*Proof of concept with LacZ alpha fragment fused to "Synthetic Import Domain" from Colicin D<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Aim :'''<br />
Harness bacteria-containing gel beads to assure cell containment and complement activity of genetic safety systems.<br />
<br />
'''Experimental system:'''<br />
Bacterial cells are encapsulated in alginate beads. We used a [https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation#Cell_Containment_Assay cell containment assay] based on plating to assess the release of cells from alginate beads. In addition, we aimed at improving the entrapment of cells through stabilization by polyethyleneimine and covalent cross-linkage by glutaraldehyde. <br />
<br />
'''Achievements :'''<br />
*Encapsulated cells achieved and their ability to propagate and express proteins within alginate beads demonstrated.<br />
*Stabilized alginate beads by covalent cross-linkage achieved and their ability to entrap cells demonstrated.<br />
*we performed [https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation#Bristol_2010_Nitrate_Reporter additional characterization] of the Bristol 2010 nitrate reporter [http://partsregistry.org/Part:BBa_K381001 K381001]<br />
* Efficient killing by colicin producing cells was achieved within the beads.<br />
</td><br />
</tr><br />
</table><br />
<br />
==Human Practice==<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b>Aim</b><br />
<br />
To chart new venues of best practice for synthetic biology. To this end, we examined the ethical, biological and social concerns related to the release of genetically modified bacteria in the wild.<br />
<br />
<b>Metodology</b><br />
<br />
#'''''Interviews with experts''''' which enabled us to have a broad overview of the state of the art. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Interview Read More]<br />
#'''''Interaction with high-schoolers''''' to have first-hand appreciation of reactions from first exposure to synthetic biology<br />
#'''''We screened previous iGEM team’s wikis''''' to trace the evolution of biosafety concerns and devices in the iGEM community, focusing on proposed containment systems. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/WikiScreen Read More]<br />
#'''''We focused on horizontal gene transfer as main generic risk factor'''''. <br />
#'''''Synthetic report''''' where we addressed the concerns raised by synthetic biology per se, that is, as a technique. Then, we analyzed the specific concerns that arise from synthetic biology’s potential applications in nature. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report Read More]<br />
<br />
<br />
<b>Main Conclusions</b><br />
# Societal interaction: <br />
#:*'''''The need to raise awareness''''' of synthetic biology in the population so people can decide in the most enlightened way possible if they want of this new technology and of its applications (A),<br />
#:* '''''The need of a discussion''''' between society’s different protagonists to set goals, define what they would consider as benefits and acceptable risks (B),<br />
# Best research practice:<br />
#:* '''''Zero risk is impossible to achieve''''' as no containment system can be 100% safe (bacteria can always escape by mutations) (C), <br />
#:* There is a '''''lack of quantitative data evaluating the probability of failure of any synthetic biology engineered system, in particular containment systems''''' (D),<br />
#:* There is a '''''lack of quantitative data evaluating the risk of HGT assuming containment systems failed''''' (E),<br />
#:* The compiling of the wiki screen shows that '''''no containment systems created in iGEM is robust''''': they lack the above quantification and are mostly one mutation away from failure. We call for major effort of the iGEM community to quantify available containment systems and search for new solutions (F),<br />
#:* '''''The need for an INDEPENDENT cohort of scientists''''' to test experimentally any application of synthetic biology that requires releasing in the environment (G), <br />
You can find the full list of conclusions [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report here]<br />
<br />
<b>Main Proposals</b><br />
# Societal interaction: <br />
#:* '''''Organizing a workshop''''' on synthetic biology and a tour of our lab for 60 high school students, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Workshop Read More]. First initiative for teaching synthetic biology in French high-school leading to a high-school iGEM team. Ultimately, we would like interaction with high school or middle school students to be a requirement for an iGEM gold medal. <br />
#:* '''''Organizing a debate''''' with 10 non expert students from various background, and then opening the debate to the floor (the public), which was made up of both experts and non experts, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Debate Read More]. <br />
#:* '''''Creating a page to explain horizontal gene transfer''''' to non scientists. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/HGT Go to HGT page]<br />
# Best research practice:<br />
#:* '''''Creating a system as robust as possible''''', that is many mutations away from failure (this is what our [https://2012.igem.org/Team:Paris_Bettencourt/Overview bench work] has been all about) (addresses issue C and F),<br />
#:* '''''Creating a safety page on the biobrick registry''''' where all the safety devices that exist are listed and characterized (included evaluation of their robustness) in order for iGEM teams to pick the most appropriate device to add to their newly created genetic circuit. Ultimately, we would like '''''the integration of safety modules and risks assessments to be part of of every synthetic biology project from the very start''''' (already listed in the safety page or created de novo by the team) (addresses issue D, F), [http://partsregistry.org/Biosafety Go to safety page]<br />
#:*The community has to '''''build a collection of bio-safety devices for future engineers'''''<br />
#:*Each synthetic biology application should '''''assess and disclose a list of application-specific risks and hazards'''''.<br />
#:*Development and adoption of a '''''safety chasis for synthetic biology research and prototyping'''''.<br />
<br />
You can find the full list of proposals [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report#III_Proposals here]<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/AchievementsTeam:Paris Bettencourt/Achievements2012-09-27T02:35:20Z<p>Aleksandra: /* Human Practice */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<br />
<div id="grouptitle">Achievements</div><br />
<br />
==Achievements of all the different modules==<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Semantic containment'''<br />
<br />
'''Aims :'''<br />
<br />
Creating a semantic containment system to prevent gene expression in natural organisms<br />
Characterize the system<br />
Use this system in all genes of the system, the critical genes first (e.g. colicin)<br />
System<br />
<br />
An amber codon (stop codon) embedded in protein genes to prevent their expression and an amber suppressor system in our genetically engineered bacteria<br />
<br />
'''Achievements :'''<br />
<br />
Construction and characterization of 2 biobricks :<br />
K914000 : PLac-supD-T : tRNA amber suppressor<br />
K914009 : P1003* Ser133->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
Both part were well characterized and works well. For the second parts, we show that as expected, one mutation is quite leaky, although it works qualitatively, but one mutation is not enough if we want to release such parts in nature. Other reasons emphasize this observation, notably the weakness of being at one mutation to recover the protein functionality.<br />
<br />
Creation of a new category in the part registry : Semantic containment. The aim of this category is to let people improving each part by adding for instance other amber mutations to existing part to increase the containment.<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Suicide system'''<br />
<br />
'''Aims :'''<br />
Implement a kill-switch that features population-level suicide and complete genome degradation. <br />
<br />
'''System :'''<br />
A synthetic toxin-anti-toxin system based on the wild type Colicin E2 operon.<br />
<br />
'''Achievements :'''<br />
We showed that Colicin E2 cells induce cell death in sensitive populations, and that these sensitive populations can be protected by providing them with our engineered immunity protein. <br />
* Construction of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914001 K914001] : pLac-repressilator RBS-Colicin E2 immunity protein<br />
** [http://partsregistry.org/Part:BBa_K914002 K914002] :repressilator RBS-Colicin E2 immunity protein<br />
Part K914001 is well characterized and provides immunity to sensitive cells against the Colicin E2 activity protein, but is leaky. Part K914002 is promoterless and allows users to easily plug in the appropriate promoter for their desired purpose. <br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety XNase]. The aim of this category is to provide users with DNase/RNase parts that can be used for improved kill switches featuring the degradation of genomic material.<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Restriction Enzyme System </b> <br />
<br />
<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
</tr><br />
</table><br />
<br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
'''MAGE'''<br />
<br />
'''Aims :'''<br />
<br />
Removal of four FseI restriction sites from E. coli MG1655 genome.<br />
<br />
'''Experimental System'''<br />
<br />
Using multiplex automated genome engineering (MAGE) - a technique capable of editing the genome by making small changes in existing genomic sequences.<br />
<br />
'''Achievements'''<br />
<br />
Proof of concept by introducing a stop codon in the middle of the lacZ gene<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
'''Synthetic Import Domain'''<br />
'''Aim :'''<br />
<br />
Creation of a novel protein import mechanism in bacteria. <br />
<br />
<br />
'''Experimental System'''<br />
<br />
Exploit the natural Colicin import domain fused to any protein at will, dubbed here: "Synthetic Import Domain".<br />
<br />
'''Achievements'''<br />
<br />
*Construction of colicin-like toxin by fusing Colicin E2 based "Synthetic Import Domain" with RNAse domain of colicin D<br />
*Constructon of FseI, I-SceI, LuxR active fragment, LacZ alpha fragment, PyrF and T7 RNA polymerase fused to the two types of "Synthetic Import Domains" from Colicin E2 and Colicin D<br />
*Proof of concept with LacZ alpha fragment fused to "Synthetic Import Domain" from Colicin D<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Aim :'''<br />
Harness bacteria-containing gel beads to assure cell containment and complement activity of genetic safety systems.<br />
<br />
'''Experimental system:'''<br />
Bacterial cells are encapsulated in alginate beads. We used a [https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation#Cell_Containment_Assay cell containment assay] based on plating to assess the release of cells from alginate beads. In addition, we aimed at improving the entrapment of cells through stabilization by polyethyleneimine and covalent cross-linkage by glutaraldehyde. <br />
<br />
'''Achievements :'''<br />
*Encapsulated cells achieved and their ability to propagate and express proteins within alginate beads demonstrated.<br />
*Stabilized alginate beads by covalent cross-linkage achieved and their ability to entrap cells demonstrated.<br />
*we performed [https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation#Bristol_2010_Nitrate_Reporter additional characterization] of the Bristol 2010 nitrate reporter [http://partsregistry.org/Part:BBa_K381001 K381001]<br />
* Efficient killing by colicin producing cells was achieved within the beads.<br />
</td><br />
</tr><br />
</table><br />
<br />
==Human Practice==<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b>Aim</b><br />
<br />
To chart new venues of best practice for synthetic biology. To this end, we examined the ethical, biological and social concerns related to the release of genetically modified bacteria in the wild.<br />
<br />
<b>Metodology</b><br />
<br />
#'''''Interviews with experts''''' which enabled us to have a broad overview of the state of the art. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Interview Read More]<br />
#'''''Interaction with high-schoolers''''' to have first-hand appreciation of reactions from first exposure to synthetic biology<br />
#'''''We screened previous iGEM team’s wikis''''' to trace the evolution of biosafety concerns and devices in the iGEM community, focusing on proposed containment systems. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/WikiScreen Read More]<br />
#'''''We focused on horizontal gene transfer as main generic risk factor'''''. <br />
#'''''Synthetic report''''' where we addressed the concerns raised by synthetic biology per se, that is, as a technique. Then, we analyzed the specific concerns that arise from synthetic biology’s potential applications in nature. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report Read More]<br />
<br />
<br />
<b>Main Conclusions</b><br />
# Societal interaction: <br />
#:*'''''The need to raise awareness''''' of synthetic biology in the population so people can decide in the most enlightened way possible if they want of this new technology and of its applications (A),<br />
#:* '''''The need of a discussion''''' between society’s different protagonists to set goals, define what they would consider as benefits and acceptable risks (B),<br />
# Best research practice:<br />
#:* '''''Zero risk is impossible to achieve''''' as no containment system can be 100% safe (bacteria can always escape by mutations) (C), <br />
#:* There is a '''''lack of quantitative data evaluating the probability of failure of any synthetic biology engineered system, in particular containment systems''''' (D),<br />
#:* There is a '''''lack of quantitative data evaluating the risk of HGT assuming containment systems failed''''' (E),<br />
#:* The compiling of the wiki screen shows that '''''no containment systems created in iGEM is robust''''': they lack the above quantification and are mostly one mutation away from failure. We call for major effort of the iGEM community to quantify available containment systems and search for new solutions (F),<br />
#:* '''''The need for an INDEPENDENT cohort of scientists''''' to test experimentally any application of synthetic biology that requires releasing in the environment (G), <br />
You can find the full list of conclusions [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report here]<br />
<br />
<b>Main Proposals</b><br />
# Societal interaction: <br />
#:* '''''Organizing a workshop''''' on synthetic biology and a tour of our lab for 60 high school students, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Workshop Read More]. First initiative for teaching synthetic biology in French high-school leading to a high-school iGEM team. Ultimately, we would like interaction with high school or middle school students to be a requirement for an iGEM gold medal. <br />
#:* '''''Organizing a debate''''' with 10 non expert students from various background, and then opening the debate to the floor (the public), which was made up of both experts and non experts, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Debate Read More]. <br />
#:* '''''Creating a page to explain horizontal gene transfer''''' to non scientists. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/HGT Go to HGT page]<br />
# Best research practice:<br />
#:* '''''Creating a system as robust as possible''''', that is many mutations away from failure (this is what our [https://2012.igem.org/Team:Paris_Bettencourt/Overview bench work] has been all about) (addresses issue C and F),<br />
#:* '''''Creating a safety page on the biobrick registry''''' where all the safety devices that exist are listed and characterized (included evaluation of their robustness) in order for iGEM teams to pick the most appropriate device to add to their newly created genetic circuit. Ultimately, we would like '''''the integration of safety modules and risks assessments to be part of of every synthetic biology project from the very start''''' (already listed in the safety page or created de novo by the team) (addresses issue D, F), [http://partsregistry.org/Biosafety Go to safety page]<br />
#:*The community has to '''''build a collection of bio-safety devices for future engineers'''''<br />
#:*Each synthetic biology application should '''''assess and disclose a list of application-specific risks and hazards'''''.<br />
#:*Development and adoption of a '''''safety chasis for synthetic biology research and prototyping'''''.<br />
<br />
You can find the full list of proposals [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report#III_Proposals here]<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
==Human Practice==<br />
<br />
</td><br />
</tr><br />
</table><br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/AchievementsTeam:Paris Bettencourt/Achievements2012-09-27T02:35:05Z<p>Aleksandra: /* Human Practice */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<br />
<div id="grouptitle">Achievements</div><br />
<br />
==Achievements of all the different modules==<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Semantic containment'''<br />
<br />
'''Aims :'''<br />
<br />
Creating a semantic containment system to prevent gene expression in natural organisms<br />
Characterize the system<br />
Use this system in all genes of the system, the critical genes first (e.g. colicin)<br />
System<br />
<br />
An amber codon (stop codon) embedded in protein genes to prevent their expression and an amber suppressor system in our genetically engineered bacteria<br />
<br />
'''Achievements :'''<br />
<br />
Construction and characterization of 2 biobricks :<br />
K914000 : PLac-supD-T : tRNA amber suppressor<br />
K914009 : P1003* Ser133->Amber Codon : kanamycin gene resistance with 1 amber mutation<br />
Both part were well characterized and works well. For the second parts, we show that as expected, one mutation is quite leaky, although it works qualitatively, but one mutation is not enough if we want to release such parts in nature. Other reasons emphasize this observation, notably the weakness of being at one mutation to recover the protein functionality.<br />
<br />
Creation of a new category in the part registry : Semantic containment. The aim of this category is to let people improving each part by adding for instance other amber mutations to existing part to increase the containment.<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Suicide system'''<br />
<br />
'''Aims :'''<br />
Implement a kill-switch that features population-level suicide and complete genome degradation. <br />
<br />
'''System :'''<br />
A synthetic toxin-anti-toxin system based on the wild type Colicin E2 operon.<br />
<br />
'''Achievements :'''<br />
We showed that Colicin E2 cells induce cell death in sensitive populations, and that these sensitive populations can be protected by providing them with our engineered immunity protein. <br />
* Construction of 2 biobricks :<br />
** [http://partsregistry.org/Part:BBa_K914001 K914001] : pLac-repressilator RBS-Colicin E2 immunity protein<br />
** [http://partsregistry.org/Part:BBa_K914002 K914002] :repressilator RBS-Colicin E2 immunity protein<br />
Part K914001 is well characterized and provides immunity to sensitive cells against the Colicin E2 activity protein, but is leaky. Part K914002 is promoterless and allows users to easily plug in the appropriate promoter for their desired purpose. <br />
* Creation of a new category in the part registry : [http://partsregistry.org/Biosafety XNase]. The aim of this category is to provide users with DNase/RNase parts that can be used for improved kill switches featuring the degradation of genomic material.<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Restriction Enzyme System </b> <br />
<br />
<b> Aim: </b><br />
<br />
To design a plasmid self-digestion system.<br />
<br />
<b>Experimental System:</b> <br />
<br />
We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Measuring_the_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterization_of_pRha Characterization ] of the L-rhamnose-inducible promoter ([https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design pRha]). <br />
</tr><br />
</table><br />
<br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
'''MAGE'''<br />
<br />
'''Aims :'''<br />
<br />
Removal of four FseI restriction sites from E. coli MG1655 genome.<br />
<br />
'''Experimental System'''<br />
<br />
Using multiplex automated genome engineering (MAGE) - a technique capable of editing the genome by making small changes in existing genomic sequences.<br />
<br />
'''Achievements'''<br />
<br />
Proof of concept by introducing a stop codon in the middle of the lacZ gene<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
'''Synthetic Import Domain'''<br />
'''Aim :'''<br />
<br />
Creation of a novel protein import mechanism in bacteria. <br />
<br />
<br />
'''Experimental System'''<br />
<br />
Exploit the natural Colicin import domain fused to any protein at will, dubbed here: "Synthetic Import Domain".<br />
<br />
'''Achievements'''<br />
<br />
*Construction of colicin-like toxin by fusing Colicin E2 based "Synthetic Import Domain" with RNAse domain of colicin D<br />
*Constructon of FseI, I-SceI, LuxR active fragment, LacZ alpha fragment, PyrF and T7 RNA polymerase fused to the two types of "Synthetic Import Domains" from Colicin E2 and Colicin D<br />
*Proof of concept with LacZ alpha fragment fused to "Synthetic Import Domain" from Colicin D<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
'''Aim :'''<br />
Harness bacteria-containing gel beads to assure cell containment and complement activity of genetic safety systems.<br />
<br />
'''Experimental system:'''<br />
Bacterial cells are encapsulated in alginate beads. We used a [https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation#Cell_Containment_Assay cell containment assay] based on plating to assess the release of cells from alginate beads. In addition, we aimed at improving the entrapment of cells through stabilization by polyethyleneimine and covalent cross-linkage by glutaraldehyde. <br />
<br />
'''Achievements :'''<br />
*Encapsulated cells achieved and their ability to propagate and express proteins within alginate beads demonstrated.<br />
*Stabilized alginate beads by covalent cross-linkage achieved and their ability to entrap cells demonstrated.<br />
*we performed [https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation#Bristol_2010_Nitrate_Reporter additional characterization] of the Bristol 2010 nitrate reporter [http://partsregistry.org/Part:BBa_K381001 K381001]<br />
* Efficient killing by colicin producing cells was achieved within the beads.<br />
</td><br />
</tr><br />
</table><br />
<br />
==Human Practice==<br />
<div id="grouptitle">Human Practice </div><br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b>Aim</b><br />
<br />
To chart new venues of best practice for synthetic biology. To this end, we examined the ethical, biological and social concerns related to the release of genetically modified bacteria in the wild.<br />
<br />
<b>Metodology</b><br />
<br />
#'''''Interviews with experts''''' which enabled us to have a broad overview of the state of the art. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Interview Read More]<br />
#'''''Interaction with high-schoolers''''' to have first-hand appreciation of reactions from first exposure to synthetic biology<br />
#'''''We screened previous iGEM team’s wikis''''' to trace the evolution of biosafety concerns and devices in the iGEM community, focusing on proposed containment systems. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/WikiScreen Read More]<br />
#'''''We focused on horizontal gene transfer as main generic risk factor'''''. <br />
#'''''Synthetic report''''' where we addressed the concerns raised by synthetic biology per se, that is, as a technique. Then, we analyzed the specific concerns that arise from synthetic biology’s potential applications in nature. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report Read More]<br />
<br />
<br />
<b>Main Conclusions</b><br />
# Societal interaction: <br />
#:*'''''The need to raise awareness''''' of synthetic biology in the population so people can decide in the most enlightened way possible if they want of this new technology and of its applications (A),<br />
#:* '''''The need of a discussion''''' between society’s different protagonists to set goals, define what they would consider as benefits and acceptable risks (B),<br />
# Best research practice:<br />
#:* '''''Zero risk is impossible to achieve''''' as no containment system can be 100% safe (bacteria can always escape by mutations) (C), <br />
#:* There is a '''''lack of quantitative data evaluating the probability of failure of any synthetic biology engineered system, in particular containment systems''''' (D),<br />
#:* There is a '''''lack of quantitative data evaluating the risk of HGT assuming containment systems failed''''' (E),<br />
#:* The compiling of the wiki screen shows that '''''no containment systems created in iGEM is robust''''': they lack the above quantification and are mostly one mutation away from failure. We call for major effort of the iGEM community to quantify available containment systems and search for new solutions (F),<br />
#:* '''''The need for an INDEPENDENT cohort of scientists''''' to test experimentally any application of synthetic biology that requires releasing in the environment (G), <br />
You can find the full list of conclusions [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report here]<br />
<br />
<b>Main Proposals</b><br />
# Societal interaction: <br />
#:* '''''Organizing a workshop''''' on synthetic biology and a tour of our lab for 60 high school students, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Workshop Read More]. First initiative for teaching synthetic biology in French high-school leading to a high-school iGEM team. Ultimately, we would like interaction with high school or middle school students to be a requirement for an iGEM gold medal. <br />
#:* '''''Organizing a debate''''' with 10 non expert students from various background, and then opening the debate to the floor (the public), which was made up of both experts and non experts, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Debate Read More]. <br />
#:* '''''Creating a page to explain horizontal gene transfer''''' to non scientists. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/HGT Go to HGT page]<br />
# Best research practice:<br />
#:* '''''Creating a system as robust as possible''''', that is many mutations away from failure (this is what our [https://2012.igem.org/Team:Paris_Bettencourt/Overview bench work] has been all about) (addresses issue C and F),<br />
#:* '''''Creating a safety page on the biobrick registry''''' where all the safety devices that exist are listed and characterized (included evaluation of their robustness) in order for iGEM teams to pick the most appropriate device to add to their newly created genetic circuit. Ultimately, we would like '''''the integration of safety modules and risks assessments to be part of of every synthetic biology project from the very start''''' (already listed in the safety page or created de novo by the team) (addresses issue D, F), [http://partsregistry.org/Biosafety Go to safety page]<br />
#:*The community has to '''''build a collection of bio-safety devices for future engineers'''''<br />
#:*Each synthetic biology application should '''''assess and disclose a list of application-specific risks and hazards'''''.<br />
#:*Development and adoption of a '''''safety chasis for synthetic biology research and prototyping'''''.<br />
<br />
You can find the full list of proposals [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report#III_Proposals here]<br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
==Human Practice==<br />
<br />
</td><br />
</tr><br />
</table><br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/OverviewTeam:Paris Bettencourt/Human Practice/Overview2012-09-27T02:32:23Z<p>Aleksandra: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<div id="grouptitle">Human Practice </div><br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b>Aim</b><br />
<br />
To chart new venues of best practice for synthetic biology. To this end, we examined the ethical, biological and social concerns related to the release of genetically modified bacteria in the wild.<br />
<br />
<b>Metodology</b><br />
<br />
#'''''Interviews with experts''''' which enabled us to have a broad overview of the state of the art. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Interview Read More]<br />
#'''''Interaction with high-schoolers''''' to have first-hand appreciation of reactions from first exposure to synthetic biology<br />
#'''''We screened previous iGEM team’s wikis''''' to trace the evolution of biosafety concerns and devices in the iGEM community, focusing on proposed containment systems. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/WikiScreen Read More]<br />
#'''''We focused on horizontal gene transfer as main generic risk factor'''''. <br />
#'''''Synthetic report''''' where we addressed the concerns raised by synthetic biology per se, that is, as a technique. Then, we analyzed the specific concerns that arise from synthetic biology’s potential applications in nature. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report Read More]<br />
<br />
<br />
<b>Main Conclusions</b><br />
# Societal interaction: <br />
#:*'''''The need to raise awareness''''' of synthetic biology in the population so people can decide in the most enlightened way possible if they want of this new technology and of its applications (A),<br />
#:* '''''The need of a discussion''''' between society’s different protagonists to set goals, define what they would consider as benefits and acceptable risks (B),<br />
# Best research practice:<br />
#:* '''''Zero risk is impossible to achieve''''' as no containment system can be 100% safe (bacteria can always escape by mutations) (C), <br />
#:* There is a '''''lack of quantitative data evaluating the probability of failure of any synthetic biology engineered system, in particular containment systems''''' (D),<br />
#:* There is a '''''lack of quantitative data evaluating the risk of HGT assuming containment systems failed''''' (E),<br />
#:* The compiling of the wiki screen shows that '''''no containment systems created in iGEM is robust''''': they lack the above quantification and are mostly one mutation away from failure. We call for major effort of the iGEM community to quantify available containment systems and search for new solutions (F),<br />
#:* '''''The need for an INDEPENDENT cohort of scientists''''' to test experimentally any application of synthetic biology that requires releasing in the environment (G), <br />
You can find the full list of conclusions [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report here]<br />
<br />
<b>Main Proposals</b><br />
# Societal interaction: <br />
#:* '''''Organizing a workshop''''' on synthetic biology and a tour of our lab for 60 high school students, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Workshop Read More]. First initiative for teaching synthetic biology in French high-school leading to a high-school iGEM team. Ultimately, we would like interaction with high school or middle school students to be a requirement for an iGEM gold medal. <br />
#:* '''''Organizing a debate''''' with 10 non expert students from various background, and then opening the debate to the floor (the public), which was made up of both experts and non experts, (addresses issue A and B) [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Debate Read More]. <br />
#:* '''''Creating a page to explain horizontal gene transfer''''' to non scientists. [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/HGT Go to HGT page]<br />
# Best research practice:<br />
#:* '''''Creating a system as robust as possible''''', that is many mutations away from failure (this is what our [https://2012.igem.org/Team:Paris_Bettencourt/Overview bench work] has been all about) (addresses issue C and F),<br />
#:* '''''Creating a safety page on the biobrick registry''''' where all the safety devices that exist are listed and characterized (included evaluation of their robustness) in order for iGEM teams to pick the most appropriate device to add to their newly created genetic circuit. Ultimately, we would like '''''the integration of safety modules and risks assessments to be part of of every synthetic biology project from the very start''''' (already listed in the safety page or created de novo by the team) (addresses issue D, F), [http://partsregistry.org/Biosafety Go to safety page]<br />
#:*The community has to '''''build a collection of bio-safety devices for future engineers'''''<br />
#:*Each synthetic biology application should '''''assess and disclose a list of application-specific risks and hazards'''''.<br />
#:*Development and adoption of a '''''safety chasis for synthetic biology research and prototyping'''''.<br />
<br />
You can find the full list of proposals [https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Report#III_Proposals here]<br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/e/e3/PhysicalContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/f/f6/PhysicalContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV"><b>1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth</b> </div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="https://static.igem.org/mediawiki/2012/0/09/SemanticContainmentSystemLarge.png" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br><b>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. </b></div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
<b>3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.</b><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/5/5c/RestrictionSystem.png" width="60px" border="0" /><span><img src="/wiki/images/9/97/Restriction2PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
<b>4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.</b></div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/7/78/ToxinAntitoxin.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells, while permitting their growth.<br><br>2) Semantic Containment: Our synthetic system will have an amber codon embedded in its genes. The amber suppressor system will ensure their expression in our bacteria, while preventing it in natural populations. <br><br><br />
3) Delay system: In the presence of Arabinose, LacI is produced, repressing the expression of a restriction enzyme. Once Arabinose is not present any more, the LacI repressor concentration decreases with dilution and degradation. This leads to the expression of the restriction enzyme.<br><br><br />
4) Restriction Enzyme system: The restriction enzyme destroys the plasmid carrying the synthetic circuit and the anti-toxin gene.<br><br><b>5) Suicide system: Once the anti-toxin concentration is below a given threshold, the toxin is no longer inhibited. It kills the cell as well as its neighbors, and eliminates extracellular DNA via its DNase activity.</b></div></span></a><br />
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==An example of an application for our project==<br />
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Imagine a farmer that would like to know how much fertilizer is in his field, and optimize its use. We would provide him with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in beads containing arabinose. He would spray the beads in his field, wait for 12h and then check if they are glowing in response to the nitrates contained in his soil. <br />
<br>We want to prevent the engineered organism or its DNA from being released and potentially transferred to a soil organism. For this reason once the arabinose is completely degraded inside the beads, the delay system would trigger the degradation of any DNA, followed by the collective death of our organisms due to the activation of toxic Colicins. Moreover, the semantic containment system would ensure that even if a synthetic gene is transferred to a natural organism, it would not be translated into a functional protein.<br />
Using our mechanism, the farmer would be able to use this device without endangering the environment by the release of synthetic genes.<br />
<br />
==Objectives==<br />
<br />
Our project aims to:<br />
<br />
*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
<br />
To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
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====General recommandation for a good killswitch device====<br />
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*bla<br />
*blo<br />
*blu<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T01:12:09Z<p>Aleksandra: /* Recovery in glucose */</p>
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<b> Aim: </b> To design a plasmid self-digestion system.<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* Currently we are in process of [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design L-rhamnose-inducible promoter (pRha)] caracterisation. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterisation_of_pRha &#091;Read more&#093;]<br />
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==Overview==<br />
Our group was responsible for designing a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid, including the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. Afterwards the cell's DNA can be degraded by the Colicin.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*1010 base pairs or once in 20 human genomes.</li><br />
</ol><br />
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<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
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<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
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</li><br />
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<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
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<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
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==Experiments and results==<br />
<br />
===Measuring the efficiency of I-SceI (Cloned parts)===<br />
To measure the digestion efficiency of I-SceI, we did a trasformation of two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was sent us by [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
Firstely, we decided to check the first hypothesis, and to check if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|Selection: Chloramphenicol]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|Selection: Ampicillin]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|Selection: Chloramphenicol & Ampicillin]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|Selection: Chloramphenicol]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|Selection: Ampicillin]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|Selection: Chloramphenicol & Ampicillin]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|Selection: Chloramphenicol]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|Selection: Ampicillin]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|Selection: Chloramphenicol & Ampicillin]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
<br />
<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Present your results<br />
<br />
===Characterisation of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|-<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
<br />
{|align="center"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T01:09:31Z<p>Aleksandra: /* Photos of fluorescence: */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b> To design a plasmid self-digestion system.<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* Currently we are in process of [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design L-rhamnose-inducible promoter (pRha)] caracterisation. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterisation_of_pRha &#091;Read more&#093;]<br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
Our group was responsible for designing a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid, including the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. Afterwards the cell's DNA can be degraded by the Colicin.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*1010 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===Measuring the efficiency of I-SceI (Cloned parts)===<br />
To measure the digestion efficiency of I-SceI, we did a trasformation of two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was sent us by [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
Firstely, we decided to check the first hypothesis, and to check if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
<br />
<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Present your results<br />
<br />
===Characterisation of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|-<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
<br />
{|align="center"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T01:09:13Z<p>Aleksandra: /* Day light photos: */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b> To design a plasmid self-digestion system.<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* Currently we are in process of [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design L-rhamnose-inducible promoter (pRha)] caracterisation. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterisation_of_pRha &#091;Read more&#093;]<br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
Our group was responsible for designing a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid, including the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. Afterwards the cell's DNA can be degraded by the Colicin.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*1010 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===Measuring the efficiency of I-SceI (Cloned parts)===<br />
To measure the digestion efficiency of I-SceI, we did a trasformation of two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was sent us by [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
Firstely, we decided to check the first hypothesis, and to check if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
<br />
<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Present your results<br />
<br />
===Characterisation of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|-<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
<br />
{|align="center"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T01:08:08Z<p>Aleksandra: /* Results */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
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<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b> To design a plasmid self-digestion system.<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* Currently we are in process of [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design L-rhamnose-inducible promoter (pRha)] caracterisation. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterisation_of_pRha &#091;Read more&#093;]<br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
Our group was responsible for designing a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid, including the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. Afterwards the cell's DNA can be degraded by the Colicin.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*1010 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===Measuring the efficiency of I-SceI (Cloned parts)===<br />
To measure the digestion efficiency of I-SceI, we did a trasformation of two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was sent us by [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
Firstely, we decided to check the first hypothesis, and to check if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
<br />
<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Present your results<br />
<br />
===Characterisation of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
Both photos are taken after we centrifuged a culture of NEB Turbo strain with transformed plasmid. For the fluorescent result, the same tubes were photographed under excitation light (540nm), through an emission filter (590nm). <br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> <b>The right tube</b> which was induced by L-rhamnose expresses RFP, while <b>the left tube</b> where we didn't add it, has no visible expression.</font>]]<br />
|-<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
<br />
{|align="center"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Quantification of the fluorescence after 10h of growth</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T01:04:28Z<p>Aleksandra: /* Characterisation of pRha */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
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<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b> To design a plasmid self-digestion system.<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* Currently we are in process of [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design L-rhamnose-inducible promoter (pRha)] caracterisation. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterisation_of_pRha &#091;Read more&#093;]<br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
Our group was responsible for designing a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid, including the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. Afterwards the cell's DNA can be degraded by the Colicin.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*1010 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===Measuring the efficiency of I-SceI (Cloned parts)===<br />
To measure the digestion efficiency of I-SceI, we did a trasformation of two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was sent us by [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
Firstely, we decided to check the first hypothesis, and to check if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
<br />
<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Present your results<br />
<br />
===Characterisation of pRha===<br />
<br />
We have submitted to the registry a new characterized promoter: pRha [http://partsregistry.org/wiki/index.php?title=Part:BBa_K914003 K914003].<br />
<br />
<br />
====Experimental setup====<br />
In order to characterize this promoter, we made a construct with a medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and an RFP ([]) cloned downstream of the pRha, on the pSB3C5 plasmid. We induced the expression of RFP by adding L-Rhamnose. As a negative control, we used cells without the inducer, as well as cells repressed with Glucose.<br />
<br />
====Results====<br />
First, by simple observation under a fluorescence viewer, we have seen that the addition of 1% L-Rhamnose leads to a significant expression of RFP after 10hours. The negative controls, where no Rhamnose was added, or when the promoter was repressed by Glucose, did not show any visible fluorescence. <br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> Both photos are taken after we centrifuged a calture of NEB Turbo strain with transformed plasmid: pRha & RBS & RFP [pSB3C5]. <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> The same tubes under excitation light (540nm). Photo is taken through emission filter (590nm). We can clearly see that <b>the right tube</b> tube which was induced by L-rhamnose express RFP, while <b>the left tube</b> where we didn't add it has no expression.</font>]]<br />
|-<br />
|}<br />
<br />
We quantified this result in a plate reader.<br />
<br />
<br />
{|align="center"<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Hi</font>]]<br />
|}<br />
<br />
Next, we characterized the pRha promoter using a plate reader. We used different concentrations of L-Rhamnose (0.05%, 0.1%, 0.2%, 0.5% and 1%) and observed the resulting fluorescence over time. As negative controls, we used the non-induced cells, as well as cells repressed by 1% Glucose.<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T00:35:05Z<p>Aleksandra: /* Results */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<!-- ########## Don't edit above ########## --><br />
<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b> To design a plasmid self-digestion system.<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* Currently we are in process of [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design L-rhamnose-inducible promoter (pRha)] caracterisation. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterisation_of_pRha &#091;Read more&#093;]<br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
Our group was responsible for designing a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid, including the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. Afterwards the cell's DNA can be degraded by the Colicin.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*1010 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===Measuring the efficiency of I-SceI (Cloned parts)===<br />
To measure the digestion efficiency of I-SceI, we did a trasformation of two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was sent us by [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
Firstely, we decided to check the first hypothesis, and to check if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
<br />
<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Present your results<br />
<br />
===Characterisation of pRha===<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> Both photos are taken after we centrifuged a calture of NEB Turbo strain with transformed plasmid: pRha & RBS & RFP [pSB3C5]. <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> The same tubes under excitation light (540nm). Photo is taken through emission filter (590nm). We can clearly see that <b>the right tube</b> tube which was induced by L-rhamnose express RFP, while <b>the left tube</b> where we didn't add it has no expression.</font>]]<br />
|-<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Hi</font>]]<br />
|}<br />
<br />
<br />
<br />
====Experimental setup====<br />
Describe the experiment<br />
<br />
====Results====<br />
Present your results<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/Restriction_EnzymeTeam:Paris Bettencourt/Restriction Enzyme2012-09-27T00:34:29Z<p>Aleksandra: /* Results */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
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<br />
<div id="grouptitle">Restriction Enzyme System</div><br />
<br />
<table id="tableboxed"><br />
<tr><br />
<td><br />
<br />
<b> Aim: </b> To design a plasmid self-digestion system.<br />
<br />
'''Achievements :'''<br />
* Construction of 4 biobricks [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter<br />
** [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac<br />
** [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad<br />
** [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha<br />
* Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_efficiency_of_I-SceI_.28Cloned_parts.29 &#091;Read more&#093;]<br />
<br />
* Characterization of 2 biobricks from TUDelft [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Mesuring_of_I-SceI_efficiency_.28TUDelft_parts.29 &#091;Read more&#093;]:<br />
** [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator<br />
** [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site<br />
* Currently we are in process of [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Design L-rhamnose-inducible promoter (pRha)] caracterisation. [https://2012.igem.org/Team:Paris_Bettencourt/Restriction_Enzyme#Characterisation_of_pRha &#091;Read more&#093;]<br />
</tr><br />
</table><br />
<br />
<br />
<br />
==Overview==<br />
Our group was responsible for designing a plasmid self-digestion system. This synthetic system allows to digest plasmids into linear parts of DNA and thus disrupt the expression of the genes carried by this plasmid, including the antitoxin (Colicin immunity protein), and any plugged-in synthetic device. Afterwards the cell's DNA can be degraded by the Colicin.<br />
<br />
==Objectives==<br />
# Find appropriate restriction enzymes which have to match the following properties:<br />
#* The corresponding restriction site must not be found in the <i>E.Coli</i> genome;<br />
#* The enzyme has to have high specifity;<br />
#* It has to work in wide range of different conditions (pH, T°, etc)<br />
# Choose a strong yet tightly repressible promoter to regulate the restriction enzyme expression;<br />
# Clone circuits with different combinations of the chosen restriction enzymes and promoters;<br />
# Measure the degradation efficiency of the restriction enzyme for each circuit;<br />
# Based on the best combination, design a self-disruption plasmid.<br />
<br />
==Design==<br />
<br />
According to the first two of our objectives, we should find an appropriate restriction enzyme and to choose a strong yet tightly repressible promoter to regulate its expression.<br />
<br />
<b>Restriction enzyme candidates:</b><br />
<br />
<ol><br />
<li><b>Fse I</b> is a restriction endonuclease which recognizes an 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The reason why we chose it is because it has the lowest number of restriction sites in the <i>E.coli</i> genome: only 4 copies. We decided to use MAGE to remove those sites from the chromosome ([https://2012.igem.org/Team:Paris_Bettencourt/MAGE see for more details]). However, MAGE did not have the expected yield, and we decided to freeze the work on this restriction enzyme and focus on the second candidate.</li><br />
<br />
<li><b>I-SceI</b> is an intron-encoded endonuclease. It is present in the mitochondria of <i>Saccharomyces cerevisiae</i> and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*1010 base pairs or once in 20 human genomes.</li><br />
</ol><br />
<br />
<br/><br />
<br />
<b>Promoter candidates:</b><br />
<br />
<ol><br />
<br />
<li><b>pLac</b><br />
<br/><br />
<ul><br />
<li><br />
Standard pLac promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_R0011 R0011].<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup><br />
</li><br />
</ul><br />
</li><br />
<br />
<li><b>pBad</b><br />
<br/><br />
<ul><br />
<li>Standard pBad promoter from the Parts Registry: [http://partsregistry.org/Part:BBa_I13453 I13453].<sup>[</sup><sup>[[#References|8]]</sup><sup>]</sup></li><br />
</ul><br />
</li><br />
<br />
<li><b>pRha</b> L-rhamnose-inducible promoter is capable of high-level protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose and by the addition of D-glucose.<br />
<br />
<ul><br />
<br />
<li>pRha is probably the best candidate for us because of two reasons:<br />
<br />
<ol><br />
<li> It is a new promoter, and so it will be orthogonal to any other existing system. It supports the modularity idea of our project.</li><br />
<li> It was reported in the literature to be appropriate for the expression of toxic genes.</li><br />
</ol><br />
<br />
</li><br />
<br />
<li>L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.<sup>[</sup><sup>[[#References|7]]</sup><sup>]</sup></li><br />
<br />
[[Image:Paris_Bettencourt_2012_Prha.png|thumb|400px|right|alt=The E. coli rhaBRS locus|<font size="1"><b>The ''E. coli'' rhaBRS locus.</b> In the presence of L-rhamnose, RhaR activates transcription of ''rhaR'' and ''rhaS'', resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the ''rhaB'' promoter.</font>]]<br />
<br />
<li>The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.<sup>[</sup><sup>[[#References|11]]</sup><sup>]</sup></li><br />
<br />
<li>The pRha sequence was containing an EcoRI restriction site, so we had to disrupt it in order to use pRha as a biobrick. In order to decide which base pair to modify, we used the [http://microbes.ucsc.edu UCSC Microbial Genome Browser]. We compared the pRha sequence in E.coli and similar species, and identified that the at the position is sometimes replaced by a in some species, so we decided to replace it in a same way. We ordered a gBlock with the pRha sequence having the mutation, and this is the sequence we used and submitted.</li><br />
<br />
</ul><br />
<br />
</li><br />
</ol><br />
<br />
Considering these candidates, we decided to clone the following constructs in low-copy vector pSB3C5 to use it in our experiments, all with a medium RBS [http://partsregistry.org/Part:BBa_B0032 B0032]:<br />
<br />
<center><br />
{| class="wikitable" width="90%" style="text-align: center;"<br />
| pBad & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pBad & RBS & RFP<br />
|-<br />
| pLac & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pLac & RBS & RFP<br />
|-<br />
| pRha & RBS & I-SceI<br />
| pRha & RBS & GFP<br />
| pRha & RBS & RFP<br />
|-<br />
|}<br />
</center><br />
<br />
==Experiments and results==<br />
<br />
===Measuring the efficiency of I-SceI (Cloned parts)===<br />
To measure the digestion efficiency of I-SceI, we did a trasformation of two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: p15a<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]. This biobrick was sent us by [https://2012.igem.org/Team:TU-Delft TUDelft iGEM team].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.<br />
<br />
====Transformation results====<br />
<br />
<br />
From the experiment we can clearly see that on plates with two antibiotics (Chloramphenicol, Cm; & Ampicillin, Amp) there are no colonies, while on plates with only one antibiotic Cm or Amp there are numerous colonies.<br />
<br />
=====The first combination of plasmids:=====<br />
<br />
*First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The second combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
=====The third combination of plasmids:=====<br />
<br />
*First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br />
*Second plasmid: I-SceI restriction site &#091;Amp&#093;<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]<br />
|}<br />
<br />
We suggested <b>two hypotheses</b> to explain the results:<br />
<br />
<ol><br />
<li><b>Those two plasmids are not compatible.</b> Plasmids could have different origins of replication. That might be the reason why double transformation is unsuccessful. </li><br />
<br />
<li><b>Our system works.</b> Our system perfectly works, but there is some leakage in the promoter leading to the expression of I-SceI meganuclease. In such case, it very efficiently cuts I-SceI restriction site, digesting the second plasmid with ampicillin resistance.</li><br />
</ol><br />
<br />
<br />
Firstely, we decided to check the first hypothesis, and to check if two plasmids are compatible with each other.<br />
<br />
<br/><br />
<br />
====Control for plasmid compatibility====<br />
<br />
As control experiment, we decided to trasform two plasmids into NEB Turbo <i>E.Coli</i> strain. The first plasmid in this experiment is analogous to the one from the previous experiment, but with GFP insted of I-SceI meganuclease; the second plasmid is the same as in the previous experiment:<br />
<br />
#<b>First plasmid:</b> Low copy plasmid with encoded generator to express GFP meganucllease. Only the version with pLac promoter was tested, because they all have the same backbone plasmid, and consequently the same replication prigin.<br />
#* Backbone: pSB3C5<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027].<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
We expected to have colonies on both type of plates: firstly, on plates with one antibiotic (Chloramphenicol & Ampicillin), secondly, on plates with both antibiotics.<br />
<br />
Our expectations became true, and our cells expressed GFP, so we conclude these two plasmids have compatible replication origins, and there should be nothing preventing the I-SceI from being expressed in the previous experiment. That means that our circuits work, but there is some leaky expression of I-SceI meganuclease that leads to a very efficient digestion of the plasmid carrying the Ampicillin antibiotic. <br />
<br />
=====Day light photos:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
=====Photos of fluorescence:=====<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Amp]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_GFP_TUD_F_2.jpg|thumb|250px|center|<font size="1">Selection: Cm & Amp]]<br />
|}<br />
<br />
<br />
To avoid leakage, in the next experiment we tried to recover cells after transformation and plate it in the presence of glucose that represses the pLac promoter.<br />
<br />
<br/><br />
<br />
=====Recovery in glucose=====<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_106TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pBad & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_109TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_G.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol & Ampicillin<br/> First plasmid: pRha & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Even if we recover the double transformants in the presence of Glucose to tightly repress the expression of the meganuclease, we still have no colonies on the plates containing both Amp and Cm. This is a sign that our construct is leaky, and the expression and function of I-SceI is so efficient that it digests all plasmids containing the corresponding restriction site, and the cells are no longer resistant to Amp.<br />
We concluded that the LVA tag added by the TUDelft team is really essential to control the endonuclease expression. However, in the case of TUDelft's construct, it's not efficient enough to kill all cells, whereas in our construct, it's too efficient and kills all cells even without induction. For future, we would need to increase the expression level of the I-SceI, while decreasing the leakiness.<br />
<br />
===Measuring the I-SceI efficiency (TUDelft parts)===<br />
In 2009, [https://2009.igem.org/Team:TUDelft/SDP_Overview TUDelft iGEM Team] has already tried to design a Self Destructive Plasmid based on I-SceI meganuclease. For their experiments, they designed a generator to produce I-SceI meganuclease and used the same pLac promoter, but they had two essential differences with our system:<br />
<br />
* They used a [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030 strong RBS].<br />
* I-SceI meganuclease they used had an LVA tag.<br />
<br />
Moreover, they didn't submit the meganuclease alone as a biobrick, so it couldn't be used for constructing new composite biobricks controlled by other promoters, which would be very useful for the modularity of our system. That is why we started working on our own constructions of meganuclease first. However, we asked TUDelft to send us two plasmids that they designed, so that we can test and characterize them:<br />
<br />
#<b>First plasmid:</b> High copy plasmid with encoded generator to express I-SceI meganucllease, [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]:<br />
#* Backbone: pSB1C3<br />
#* Resistance: Chloramphenicol<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
#*: <br />
#*: <br />
#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 K175027]:<br />
#* Backbone: pSB1AK3<br />
#* Resistance: Ampicillin and Kanamycin<br />
#* Origin of Replication: modified pMB1 derived from pUC19<br />
<br />
=====Step 1=====<br />
To mesure the efficiency of I-SceI from TUDelft parts, we proceeded in the same way as for the characterization of our own designs: we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain.<br />
<br />
The transformation was successful.<br />
<br />
=====Step 2=====<br />
<br />
<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_Cm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|-<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_woIPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (no induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_AmpCm_TUD_IPTG.jpg|thumb|250px|center|<font size="1">Selection: Chloramphenicol (IPTG induced)<br/> First plasmid: pLac & RBS & I-SceI &#091;Cm&#093;<br/>Second plasmid: I-SceI restriction site &#091;Amp&#093;</font>]]<br />
|}<br />
<br />
====Results====<br />
Present your results<br />
<br />
===Characterisation of pRha===<br />
<br />
{|align="center"<br />
|-valign="top"<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_1.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> Both photos are taken after we centrifuged a calture of NEB Turbo strain with transformed plasmid: pRha & RBS & RFP [pSB3C5]. <b>The left tube:</b> cells were grown without L-rhamnose. <b>The right tube:</b> cells are grown in the present of 1% of L-rhamnose.</font>]]<br />
|[[Image:Paris_Bettencourt_2012_RG_pRha_photo_2.jpg|thumb|250px|center|<font size="1"><i>E.Coli</i> The same tubes under excitation light (540nm). Photo is taken through emission filter (590nm). We can clearly see that <b>the right tube</b> tube which was induced by L-rhamnose express RFP, while <b>the left tube</b> where we didn't add it has no expression.</font>]]<br />
|-<br />
| colspan = 2 | [[Image:Paris_Bettencourt_2012_RG_pRha_graph_2.jpg|thumb|530px|center|<font size="1">Hi</font>]]<br />
|}<br />
<br />
<br />
<br />
====Experimental setup====<br />
Describe the experiment<br />
<br />
====Results====<br />
Present your results<br />
<br />
==References==<br />
<br />
# ''&#171;Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′&#187;'', Janise Meyertons Nelson+, Sheila M. Miceli, Mary P. Lechevalier1 and Richard J. Roberts*, (1990)<br />
# ''&#171;Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV&#187;'', Wernette C. M. Biochem Biophys Res Commun, (1998)<br />
# Yisheng Kang et al., ''&#171;Systematic Mutagenesis of E.coli K-12 MG1655 ORFs&#187;'', Yisheng Kang, Tim Durfee, Jeremy D. Glasner, Yu Qiu, David Frisch, Kelly M. Winterberg, and Frederick R. Blattner, (2004)<br />
# ''&#171;On Spontaneous DNA Damage in Single Living Cells&#187;'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):<br />
# ''&#171;A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation&#187;'', Rebecca G. Ponder, Natalie C. Fonville, Susan M. Rosenberg, (2005)<br />
# ''&#171;Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease&#187;'', L. Colleaux, L. d'Auriol, M. Betermier, G. Cottarel, A. Jacquier, F. Galibert†, B. Dujon, (1986)<br />
# ''&#171;Tightly regulated vectors for the cloning and expression of toxic genes&#187;'', Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz, (2004)<br />
# ''&#171;In Vivo Induction Kinetics of the Arabinose Promoters in Escherichia coli&#187;'' , Casonya M. Johnson And Robert F. Schleif*, (1995)<br />
# ''&#171;Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system&#187;'' Matthew J. Giacalone1, Angela M. Gentile2, Brian T. Lovitt2, Neil L. Berkley2, Carl W. Gunderson1, and Mark W. Surber, (2006)<br />
# ''&#171;DNA-Dependent Renaturation of an insoluble DNA binding Protein. Identification of the RhaS Binding Site at rhaBAD&#187;'' Susan M.Egan and Robert F. Schleif, (1994)<br />
# ''&#171;Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations&#187;'' Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, (2008)<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/SafetyTeam:Paris Bettencourt/Safety2012-09-26T23:32:36Z<p>Aleksandra: /* Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering? */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="grouptitle">Safety</div><br />
===Would any of your project ideas raise safety issues in terms of : ===<br />
====Researcher safety====<br />
We only use the Bacteria Escherichia coli DH5a NEB Turbo and K12 which are common laboratory strains [http://www.openwetware.org/wiki/E._coli_genotypes|1], also considered as Level 1 Biosafety Containment agent.<br />
<br />
However, there is a number of standard lab reagents that we require for our project, that are harmful on contact. These include:<br />
*Ethidium Bromide: <br />
Acute: Hazardous when ingested or inhaled, and is an irritant of the skin and eye. Chronic: In the long term exposure can have carcinogenic, mutagenic, and teratogenic effects, and can cause developmental toxicity.<br />
*Polyethyleneimine:<br />
May be harmful if inhaled. Causes respiratory tract irritation. May be harmful if absorbed through skin. Causes skin irritation. Causes eye irritation. May be harmful if swallowed.<br />
*Glutaraldehyde:<br />
Eye: Causes eye irritation and burns. May cause permanent visual impairment. May cause chemical conjunctivitis and corneal damage. Skin: May cause skin sensitization, an allergic reaction, which becomes evident upon re-exposure to this material. May cause hives. Causes skin irritation and burns. May cause staining of the hands (brownish or tan). Ingestion: Harmful if swallowed. Causes gastrointestinal tract burns. May cause central nervous system depression, characterized by excitement, followed by headache, dizziness, drowsiness, and nausea. Advanced stages may cause collapse, unconsciousness, coma and possible death due to respiratory failure. Possible aspiration hazard. May cause lung damage.<br />
Inhalation: Harmful if inhaled. Causes chemical burns to the respiratory tract. May cause asthma and shortness of breath. May cause nausea, dizziness, and headache. Chronic: Effects may be delayed. Repeated or prolonged exposure may cause allergic reactions in sensitive individuals.<br />
<br />
====Environmental safety====<br />
Even though it is it is generally assumed that <i>E. coli</i> would be out-competed by natural strains once it is outside of a lab, we assume the fact that in case of accidental release of any GEB, it would raise safety issues because we don't know the potential effects. So any Genetically Engineered Bacteria (GEB) can be potentially dangerous if released in the environment, either on purpose or by accident. The concern about Horizontal Gene Transfer or spread of GEB lead us to develop this project to protect the environment from synthetic devices. <br />
During the work on our Biosafety system, we protected the environment from contamination by waste products: all hazardous waste was placed in the correct container (e.g. biohazard containers for biological waste such as <i>E. coli</i> colonies), autoclaved and disposed of responsibly by the university. Team members were taught proper molecular biology skills and aseptic techniques. Team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.<br />
<br />
====Public safety====<br />
Apart from the general concern about the potential harm of GEB to the Environment and Public upon release outside of the lab, none of our designs have the potential to harm the public if released by design or accident. Our lab is especially equipped for microbial manipulation and everything is done to avoid the release. Public safety is ensured, as no member of the public is permitted access within the labs unless approved by the university, and team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.<br />
However, we are conscious that as any bacterium, <i>E.coli</i> can be hazardous. For instance, all traumatic wounds, infected burns and any serious lesions can potentially be contaminated, but it is very rare. <br />
Treatment: <i>E.coli</i> can be treated with standard antibiotics, which can be prescribed by a doctor if needed.<br />
<br />
===Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?===<br />
====Did you document these issues in the Registry?====<br />
Yes, we did. As mentioned before, biosafety is the essence of our project, and we're preoccupied by the risk of dissemination of antibiotic resistance genes, as well as any other bioactive compounds such as toxins and antitoxins. We addressed this issue in the characterization of our system. For instance, we made a semantically contained version of the Kanamycin resistance gene. We are also planning to make semantically contained version of the toxin.<br />
<br />
====How did you manage to handle the safety issue?====<br />
Several mechanisms were proposed beyond the classical laboratory safety measures :<br />
* Semantic containment that avoid the expression of synthetic genes outside the GMOs.<br />
* Meganuclease restriction sites that permit the degradation of the antitoxin.<br />
* DNases that degrade GMO's genome.<br />
<br />
====How could other teams learn from your experience?====<br />
We believe that other teams should keep in mind that not only the bacteria are potentially dangerous, but also the eventual release of their DNA could endanger the environment. We suggest that starting from next years, all iGEM teams should add a biosafety part to their project, where they should eithr use one of the existing mechanisms, or design a new one, especially if their project aims at releasing bacteria into the nature. For this purpose, we created a new [http://partsregistry.org/Biosafety Biosefety page] in the Parts Registry.<br />
<br />
===Is there a local biosafety group, committee, or review board at your institution?===<br />
The work has been carried out in the laboratory of Evolutionary Systems Biology at the Molecular, Evolutive and Medical Genetics Unit (U1001, also know as TaMaRa's lab) of the French National Institute of Medical Research ([INSERM]) within the Paris Descartes University's Medical faculty. More importantly, the Biosafety officer of our unit followed our work. Both institutions have their ethical committees though no specific issue concerning our project needed to be raised.<br />
<br />
====If yes, what does your local biosafety group think about your project?====<br />
<br />
We met the chair of the ethic committee of Paris Descartes, and they advice us to keep thinking of the human practice part, while providing us some interesting thoughts. <br />
<br />
====Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?====<br />
We created [http://partsregistry.org/Biosafety a Biosafety page] on the part registry that should list all the safety mechanisms and systems available on the part registry, with the links to their description and experimental characterization. We would like to suggest next generations of iGEM teams to consult this page, to find a system that would be useful to increase the safety of their project.<br />
We also collaborated with the iGEM Grenoble team, who proposed an additional section to the description of Biobricks™ which would explain their potential danger, and the ways to assess the risk.<br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/SafetyTeam:Paris Bettencourt/Safety2012-09-26T23:30:06Z<p>Aleksandra: /* Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="grouptitle">Safety</div><br />
===Would any of your project ideas raise safety issues in terms of : ===<br />
====Researcher safety====<br />
We only use the Bacteria Escherichia coli DH5a NEB Turbo and K12 which are common laboratory strains [http://www.openwetware.org/wiki/E._coli_genotypes|1], also considered as Level 1 Biosafety Containment agent.<br />
<br />
However, there is a number of standard lab reagents that we require for our project, that are harmful on contact. These include:<br />
*Ethidium Bromide: <br />
Acute: Hazardous when ingested or inhaled, and is an irritant of the skin and eye. Chronic: In the long term exposure can have carcinogenic, mutagenic, and teratogenic effects, and can cause developmental toxicity.<br />
*Polyethyleneimine:<br />
May be harmful if inhaled. Causes respiratory tract irritation. May be harmful if absorbed through skin. Causes skin irritation. Causes eye irritation. May be harmful if swallowed.<br />
*Glutaraldehyde:<br />
Eye: Causes eye irritation and burns. May cause permanent visual impairment. May cause chemical conjunctivitis and corneal damage. Skin: May cause skin sensitization, an allergic reaction, which becomes evident upon re-exposure to this material. May cause hives. Causes skin irritation and burns. May cause staining of the hands (brownish or tan). Ingestion: Harmful if swallowed. Causes gastrointestinal tract burns. May cause central nervous system depression, characterized by excitement, followed by headache, dizziness, drowsiness, and nausea. Advanced stages may cause collapse, unconsciousness, coma and possible death due to respiratory failure. Possible aspiration hazard. May cause lung damage.<br />
Inhalation: Harmful if inhaled. Causes chemical burns to the respiratory tract. May cause asthma and shortness of breath. May cause nausea, dizziness, and headache. Chronic: Effects may be delayed. Repeated or prolonged exposure may cause allergic reactions in sensitive individuals.<br />
<br />
====Environmental safety====<br />
Even though it is it is generally assumed that <i>E. coli</i> would be out-competed by natural strains once it is outside of a lab, we assume the fact that in case of accidental release of any GEB, it would raise safety issues because we don't know the potential effects. So any Genetically Engineered Bacteria (GEB) can be potentially dangerous if released in the environment, either on purpose or by accident. The concern about Horizontal Gene Transfer or spread of GEB lead us to develop this project to protect the environment from synthetic devices. <br />
During the work on our Biosafety system, we protected the environment from contamination by waste products: all hazardous waste was placed in the correct container (e.g. biohazard containers for biological waste such as <i>E. coli</i> colonies), autoclaved and disposed of responsibly by the university. Team members were taught proper molecular biology skills and aseptic techniques. Team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.<br />
<br />
====Public safety====<br />
Apart from the general concern about the potential harm of GEB to the Environment and Public upon release outside of the lab, none of our designs have the potential to harm the public if released by design or accident. Our lab is especially equipped for microbial manipulation and everything is done to avoid the release. Public safety is ensured, as no member of the public is permitted access within the labs unless approved by the university, and team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.<br />
However, we are conscious that as any bacterium, <i>E.coli</i> can be hazardous. For instance, all traumatic wounds, infected burns and any serious lesions can potentially be contaminated, but it is very rare. <br />
Treatment: <i>E.coli</i> can be treated with standard antibiotics, which can be prescribed by a doctor if needed.<br />
<br />
===Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?===<br />
====Did you document these issues in the Registry?====<br />
Yes, we did. As mentioned before, biosafety is the essence of our project, and we're preoccupied by the risk of dissemination of antibiotic resistance genes, as well as any other bioactive compounds such as toxins and antitoxins. We addressed this issue in the characterization of our system. For instance, we made a semantically contained version of the Kanamycin resistance gene. We are also planning to make semantically contained version of the toxin.<br />
<br />
====How did you manage to handle the safety issue?====<br />
Several mechanisms were proposed beyond the classical laboratory safety measures :<br />
* Semantic containment that avoid the expression of synthetic genes outside the GMOs.<br />
* Meganuclease restriction sites that permit the degradation of the antitoxin.<br />
* DNases that degrade GMO's genome.<br />
<br />
====How could other teams learn from your experience?====<br />
We believe that other teams should keep in mind that not only the bacteria are potentially dangerous, but also the eventual release of their DNA could endanger the environment. We suggest that starting from next years, all iGEM teams should add a biosafety part to their project, where they should eithr use one of the existing mechanisms, or design a new one, especially if their project aims at releasing bacteria into the nature. For this purpose, we created a new [http://partsregistry.org/Biosafety Biosefety page] in the Parts Registry.<br />
<br />
===Is there a local biosafety group, committee, or review board at your institution?===<br />
The work has been carried out in the laboratory of Evolutionary Systems Biology at the Molecular, Evolutive and Medical Genetics Unit (U1001, also know as TaMaRa's lab) of the French National Institute of Medical Research ([INSERM]) within the Paris Descartes University's Medical faculty. More importantly, the Biosafety officer of our unit followed our work. Both institutions have their ethical committees though no specific issue concerning our project needed to be raised.<br />
<br />
====If yes, what does your local biosafety group think about your project?====<br />
<br />
We met the chair of the ethic committee of Paris Descartes, and they advice us to keep thinking of the human practice part, while providing us some interesting thoughts. <br />
<br />
====Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?====<br />
Our idea is to create a safety page on the part registry that would list all the safety mechanism and systems available on the part registry, with the links to their description and experimental characterization. We would like to suggest next generations of iGEM teams to consult this page, to find a system that would be useful to increase the safety of their project.<br />
We also collaborate with the iGEM Grenoble team, who proposed an additional section to the description of Biobricks™ which would explain their potential danger, and the ways to assess the risk.<br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/SafetyTeam:Paris Bettencourt/Safety2012-09-26T23:12:51Z<p>Aleksandra: /* Researcher safety */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="grouptitle">Safety</div><br />
===Would any of your project ideas raise safety issues in terms of : ===<br />
====Researcher safety====<br />
We only use the Bacteria Escherichia coli DH5a NEB Turbo and K12 which are common laboratory strains [http://www.openwetware.org/wiki/E._coli_genotypes|1], also considered as Level 1 Biosafety Containment agent.<br />
<br />
However, there is a number of standard lab reagents that we require for our project, that are harmful on contact. These include:<br />
*Ethidium Bromide: <br />
Acute: Hazardous when ingested or inhaled, and is an irritant of the skin and eye. Chronic: In the long term exposure can have carcinogenic, mutagenic, and teratogenic effects, and can cause developmental toxicity.<br />
*Polyethyleneimine:<br />
May be harmful if inhaled. Causes respiratory tract irritation. May be harmful if absorbed through skin. Causes skin irritation. Causes eye irritation. May be harmful if swallowed.<br />
*Glutaraldehyde:<br />
Eye: Causes eye irritation and burns. May cause permanent visual impairment. May cause chemical conjunctivitis and corneal damage. Skin: May cause skin sensitization, an allergic reaction, which becomes evident upon re-exposure to this material. May cause hives. Causes skin irritation and burns. May cause staining of the hands (brownish or tan). Ingestion: Harmful if swallowed. Causes gastrointestinal tract burns. May cause central nervous system depression, characterized by excitement, followed by headache, dizziness, drowsiness, and nausea. Advanced stages may cause collapse, unconsciousness, coma and possible death due to respiratory failure. Possible aspiration hazard. May cause lung damage.<br />
Inhalation: Harmful if inhaled. Causes chemical burns to the respiratory tract. May cause asthma and shortness of breath. May cause nausea, dizziness, and headache. Chronic: Effects may be delayed. Repeated or prolonged exposure may cause allergic reactions in sensitive individuals.<br />
<br />
====Environmental safety====<br />
Even though it is it is generally assumed that <i>E. coli</i> would be out-competed by natural strains once it is outside of a lab, we assume the fact that in case of accidental release of any GEB, it would raise safety issues because we don't know the potential effects. So any Genetically Engineered Bacteria (GEB) can be potentially dangerous if released in the environment, either on purpose or by accident. The concern about Horizontal Gene Transfer or spread of GEB lead us to develop this project to protect the environment from synthetic devices. <br />
During the work on our Biosafety system, we protected the environment from contamination by waste products: all hazardous waste was placed in the correct container (e.g. biohazard containers for biological waste such as <i>E. coli</i> colonies), autoclaved and disposed of responsibly by the university. Team members were taught proper molecular biology skills and aseptic techniques. Team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.<br />
<br />
====Public safety====<br />
Apart from the general concern about the potential harm of GEB to the Environment and Public upon release outside of the lab, none of our designs have the potential to harm the public if released by design or accident. Our lab is especially equipped for microbial manipulation and everything is done to avoid the release. Public safety is ensured, as no member of the public is permitted access within the labs unless approved by the university, and team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.<br />
However, we are conscious that as any bacterium, <i>E.coli</i> can be hazardous. For instance, all traumatic wounds, infected burns and any serious lesions can potentially be contaminated, but it is very rare. <br />
Treatment: <i>E.coli</i> can be treated with standard antibiotics, which can be prescribed by a doctor if needed.<br />
<br />
===Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,===<br />
====did you document these issues in the Registry?====<br />
Yes, we did, as mention before, biosafety is the essence of our project, and we're preoccupied by the risk of dissemination of antibiotic resistance gene as well as any bioactive compounds or antitoxin. We addressed this issue in the characterization of our system.<br />
<br />
====how did you manage to handle the safety issue?====<br />
Several mechanisms were proposed beyond the classical laboratory safety measures :<br />
* Semantic containment that avoid the expression of synthetic gene outside the GMOs.<br />
* Meganuclease restriction sites that permit the degradation of the antitoxin.<br />
* DNases that degrade GMOs genome.<br />
<br />
====How could other teams learn from your experience?====<br />
<br />
We suggest that other teams should keep in mind that not only the bacteria is potentially dangerous, but also the DNA, and one could make everything possible to add a serious safety part, mainly when iGEM project aims at releasing bacteria in Nature.<br />
<br />
===Is there a local biosafety group, committee, or review board at your institution?===<br />
The work has been carried out in the laboratory of Evolutionary Systems Biology at the Molecular, Evolutive and Medical Genetics Unit (U1001, also know as TaMaRa's lab) of the French National Institute of Medical Research ([INSERM]) within the Paris Descartes University's Medical faculty. More importantly, the Biosafety officer of our unit followed our work. Both institutions have their ethical committees though no specific issue concerning our project needed to be raised.<br />
<br />
====If yes, what does your local biosafety group think about your project?====<br />
<br />
We met the chair of the ethic committee of Paris Descartes, and they advice us to keep thinking of the human practice part, while providing us some interesting thoughts. <br />
<br />
====Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?====<br />
Our idea is to create a safety page on the part registry that would list all the safety mechanism and systems available on the part registry, with the links to their description and experimental characterization. We would like to suggest next generations of iGEM teams to consult this page, to find a system that would be useful to increase the safety of their project.<br />
We also collaborate with the iGEM Grenoble team, who proposed an additional section to the description of Biobricks™ which would explain their potential danger, and the ways to assess the risk.<br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/SafetyTeam:Paris Bettencourt/Safety2012-09-26T23:01:27Z<p>Aleksandra: /* Would any of your project ideas raise safety issues in terms of : */</p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<div id="grouptitle">Safety</div><br />
===Would any of your project ideas raise safety issues in terms of : ===<br />
====Researcher safety====<br />
We only use the Bacteria Escherichia coli DH5a NEB Turbo and K12 which are common laboratory strains [http://www.openwetware.org/wiki/E._coli_genotypes|1], also considered as Level 1 Biosafety Containment agent.<br />
<br />
However, there is a number of standard lab reagents that we require for our project, that are harmful on contact. These include:<br />
*Ethidium Bromide : <br />
Acute: Hazardous when ingested or inhaled, and is an irritant of the skin and eye. Chronic: In the long term exposure can have carcinogenic, mutagenic, and teratogenic effects, and can cause developmental toxicity.<br />
<br />
====Environmental safety====<br />
Even though it is it is generally assumed that <i>E. coli</i> would be out-competed by natural strains once it is outside of a lab, we assume the fact that in case of accidental release of any GEB, it would raise safety issues because we don't know the potential effects. So any Genetically Engineered Bacteria (GEB) can be potentially dangerous if released in the environment, either on purpose or by accident. The concern about Horizontal Gene Transfer or spread of GEB lead us to develop this project to protect the environment from synthetic devices. <br />
During the work on our Biosafety system, we protected the environment from contamination by waste products: all hazardous waste was placed in the correct container (e.g. biohazard containers for biological waste such as <i>E. coli</i> colonies), autoclaved and disposed of responsibly by the university. Team members were taught proper molecular biology skills and aseptic techniques. Team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.<br />
<br />
====Public safety====<br />
Apart from the general concern about the potential harm of GEB to the Environment and Public upon release outside of the lab, none of our designs have the potential to harm the public if released by design or accident. Our lab is especially equipped for microbial manipulation and everything is done to avoid the release. Public safety is ensured, as no member of the public is permitted access within the labs unless approved by the university, and team members followed all necessary procedures like washing their hands with disinfectant before leaving the laboratory to avoid transmitting potentially harmful material to the public/environment.<br />
However, we are conscious that as any bacterium, <i>E.coli</i> can be hazardous. For instance, all traumatic wounds, infected burns and any serious lesions can potentially be contaminated, but it is very rare. <br />
Treatment: <i>E.coli</i> can be treated with standard antibiotics, which can be prescribed by a doctor if needed.<br />
<br />
===Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,===<br />
====did you document these issues in the Registry?====<br />
Yes, we did, as mention before, biosafety is the essence of our project, and we're preoccupied by the risk of dissemination of antibiotic resistance gene as well as any bioactive compounds or antitoxin. We addressed this issue in the characterization of our system.<br />
<br />
====how did you manage to handle the safety issue?====<br />
Several mechanisms were proposed beyond the classical laboratory safety measures :<br />
* Semantic containment that avoid the expression of synthetic gene outside the GMOs.<br />
* Meganuclease restriction sites that permit the degradation of the antitoxin.<br />
* DNases that degrade GMOs genome.<br />
<br />
====How could other teams learn from your experience?====<br />
<br />
We suggest that other teams should keep in mind that not only the bacteria is potentially dangerous, but also the DNA, and one could make everything possible to add a serious safety part, mainly when iGEM project aims at releasing bacteria in Nature.<br />
<br />
===Is there a local biosafety group, committee, or review board at your institution?===<br />
The work has been carried out in the laboratory of Evolutionary Systems Biology at the Molecular, Evolutive and Medical Genetics Unit (U1001, also know as TaMaRa's lab) of the French National Institute of Medical Research ([INSERM]) within the Paris Descartes University's Medical faculty. More importantly, the Biosafety officer of our unit followed our work. Both institutions have their ethical committees though no specific issue concerning our project needed to be raised.<br />
<br />
====If yes, what does your local biosafety group think about your project?====<br />
<br />
We met the chair of the ethic committee of Paris Descartes, and they advice us to keep thinking of the human practice part, while providing us some interesting thoughts. <br />
<br />
====Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?====<br />
Our idea is to create a safety page on the part registry that would list all the safety mechanism and systems available on the part registry, with the links to their description and experimental characterization. We would like to suggest next generations of iGEM teams to consult this page, to find a system that would be useful to increase the safety of their project.<br />
We also collaborate with the iGEM Grenoble team, who proposed an additional section to the description of Biobricks™ which would explain their potential danger, and the ways to assess the risk.<br />
<br />
<br />
<!-- ########## Don't edit below ########## --><br />
{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/OverviewTeam:Paris Bettencourt/Overview2012-09-26T22:14:33Z<p>Aleksandra: </p>
<hr />
<div>{{:Team:Paris_Bettencourt/header}}<br />
<br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth<br />
2)Delay system: In presence of Arabinose, LacI is produced repressing the expression of a restriction enzyme. Once Arabinose is not anymore present, the LacI repressor concentration goes down with dilution and degradation leading to the expression of the restriction enzyme.<br />
</div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/5/5c/RestrictionSystem.png" width="60px" border="0" /><span><img src="/wiki/images/9/97/Restriction2PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1)Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth<br />
2)Delay system: In presence of Arabinose, LacI is produced repressing the expression of a restriction enzyme. Once Arabinose is not anymore present, the LacI repressor concentration goes down with dilution and degradation leading to the expression of the restriction enzyme.<br />
3)Restriction Enzyme system: The restriction enzyme destroy a plasmid carrying an anti-toxin.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/7/78/ToxinAntitoxin.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV"> 1)Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth<br />
2)Delay system: In presence of Arabinose, LacI is produced repressing the expression of a restriction enzyme. Once Arabinose is not anymore present, the LacI repressor concentration goes down with dilution and degradation leading to the expression of the restriction enzyme.<br />
3)Restriction Enzyme system: The restriction enzyme destroy a plasmid carrying an anti-toxin cassette<br />
4)Suicide system: Once the anti-toxin is below a given threshold the toxin is not anymore titrated , the toxin kills the cell, its neighbors, and eliminates extracellular DNA via its DNase activity.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px" /><br /><div id="txtOV">Semantic Containment.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth </div></span></a><br />
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<div id="grouptitle">Project Overview</div><br />
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==An example of an application for our project==<br />
<br />
Imagine a farmer that would like to know how much fertilizer is in his field, and optimize its use. We would provide him with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in beads containing arabinose. He would spray the beads in his field, wait for 12h and then check if they are glowing in response to the nitrates contained in soil. <br />
<br>Once the result is collected, we wouldn't want any synthetic organism or DNA to be released and potentially transferred to a soil organism. That's why once the arabinose is completely degraded inside the beads, the delay system would trigger the degradation of any synthetic DNA, followed by the collective death of our organisms due to the activation of toxic Colicins. Moreover, the semantic containment system would ensure that even if a synthetic gene is transferred to a natural organism, it would not be translated into a functional protein.<br />
This way, the farmer would be able to use this device without endangering the environment by the release of synthetic genes.<br />
<br />
==Objectives==<br />
<br />
Our project aims to:<br />
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*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
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To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
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<td> <br />
====General recommandation for a good killswitch device====<br />
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*bla<br />
*blo<br />
*blu<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/OverviewTeam:Paris Bettencourt/Overview2012-09-26T22:11:26Z<p>Aleksandra: /* Story telling: An example of applications for our project */</p>
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/6/6f/DelaySystem.png" width="60px" border="0" /><span><img src="/wiki/images/b/b9/Delay1PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth<br />
2)Delay system: In presence of Arabinose, LacI is produced repressing the expression of a restriction enzyme. Once Arabinose is not anymore present, the LacI repressor concentration goes down with dilution and degradation leading to the expression of the restriction enzyme.<br />
</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="/wiki/images/5/5c/RestrictionSystem.png" width="60px" border="0" /><span><img src="/wiki/images/9/97/Restriction2PB12.gif" width="500px" /><br /><div id="txtOV"><br />
1)Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth<br />
2)Delay system: In presence of Arabinose, LacI is produced repressing the expression of a restriction enzyme. Once Arabinose is not anymore present, the LacI repressor concentration goes down with dilution and degradation leading to the expression of the restriction enzyme.<br />
3)Restriction Enzyme system: The restriction enzyme destroy a plasmid carrying an anti-toxin.</div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="/wiki/images/7/78/ToxinAntitoxin.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px"/><br /><div id="txtOV"> 1)Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth<br />
2)Delay system: In presence of Arabinose, LacI is produced repressing the expression of a restriction enzyme. Once Arabinose is not anymore present, the LacI repressor concentration goes down with dilution and degradation leading to the expression of the restriction enzyme.<br />
3)Restriction Enzyme system: The restriction enzyme destroy a plasmid carrying an anti-toxin cassette<br />
4)Suicide system: Once the anti-toxin is below a given threshold the toxin is not anymore titrated , the toxin kills the cell, its neighbors, and eliminates extracellular DNA via its DNase activity.</div></span></a><br />
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<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px" /><br /><div id="txtOV">Semantic Containment.</div></span></a><br />
<br />
<a class="thumbnail" href="#thumb"><img src="https://static.igem.org/mediawiki/2012/1/1c/SemanticContainment.png" width="60px" border="0" /><span><img src="/wiki/images/8/8f/Toxin3aPB12.gif" width="500px" /><br /><div id="txtOV">1) Physical containment: Our cells will be encapsulated with Arabinose and LB in order to avoid release of DNA/cells and to permit growth </div></span></a><br />
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<div id="grouptitle">Project Overview</div><br />
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==An example of an application for our project==<br />
<br />
Imagine a farmer that would like to know how much fertilizer is in his field, and optimize its use. We would provide him with cells carrying a nitrate biosensor (AgrEcoli), encapsulated in beads containing arabinose. He would spray the beads in his field, wait for 12h and then check if they are glowing in response to the nitrates contained in soil. <br />
<br>Once the result is collected, we wouldn't want any synthetic organism or DNA to be released and potentially transferred to a soil organism. That's why once the arabinose is completely degraded inside the beads, the delay system would trigger the degradation of any synthetic DNA, followed by the collective death of our organisms due to the activation of toxic Colicins. Moreover, the semantic containment system would ensure that even if a synthetic gene is transferred to a natural organism, it would not be translated into a functional protein.<br />
This way, the farmer would be able to use this device without endangering the environment by the release of synthetic genes.<br />
<br />
==Objectives==<br />
<br />
Our project aims to:<br />
<br />
*Raise the issue of biosafety, and advocate the discerning use of biosafety circuits in future iGEM projects as a requirement<br />
*Evaluate the risk of HGT in different SynBio applications<br />
*Develop a new, improved containment system to expand the range of environments where GEOs can be used safely.<br />
<br />
To do so, we:<br />
<br />
*Engaged the general public and scientific community through debate<br />
*Raised the question about how we can regulate this practices<br />
*Compiled a parts page of safety circuits in the registry<br />
*Relied on three levels of containment :<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Encapsulation Physical containment] with alginate capsules<br />
*#[https://2012.igem.org/Team:Paris_Bettencourt/Semantic_containment Semantic containment] using an amber suppressor system<br />
*#An improved killswitch featuring [https://2012.igem.org/Team:Paris_Bettencourt/Delay delayed] population-level [https://2012.igem.org/Team:Paris_Bettencourt/Suicide#Experiments_and_results suicide] through complete genome degradation.<br />
<br />
We strived to make our system as robust against mutations as possible. <br />
<br />
<table id="tableboxed" style="border-color:rgb(176,18,31);"><br />
<tr><br />
<td> <br />
====General recommandation for a good killswitch device====<br />
<br />
*bla<br />
*blo<br />
*blu<br />
<br />
</td><br />
</tr><br />
</table><br />
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===Step 1===<br />
[[File:Paris_Bettencourt_2012_General_Circuit_s1.gif|center|550px|Step 1.]]<br />
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===Step 2===<br />
[[File:Paris_Bettencourt_2012_General_Circuit_s2.gif|center|550px|Step 2.]]<br />
Step 2.<br />
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===Step 3===<br />
[[File:Paris_Bettencourt_2012_General_Circuit_s3.gif|center|550px|Step 3.]]<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_BettencourtTeam:Paris Bettencourt2012-09-26T21:49:10Z<p>Aleksandra: </p>
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=bWARE: How Safe is Safe Enough?=<br />
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Synthetic biologists and iGEM teams in particular design Genetically Engineered Organisms (GEOs) to benefit people and communities around the world. However, many proposed applications necessarily involve the deployment of GEOs in natural environments. These dreams can never be made real without technical, legal and ethical guidelines for the use of GEOs outside the lab. Our project addresses this serious need from our perspective as safety bioengineers, citizens and humans.<br />
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<br><br> We have developed the <b>bWARE containment module</b> to substantially reduce the risk of Horizontal Gene Trasfer (HGT) while remaining compatible with existing iGEM devices. A GEO may first perform its beneficial function during a programmed delay. Then our system activates, irreversibly degrading DNA throughout the population, leaving no genetic information behind. Multiple cooperative systems provide redundancy against inevitable mutations or external stresses.<br />
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<br><br><b>Human practice considerations</b> influenced every stage of our design process. Given different biosafety modules that may be used singly or in combination, what are the best practices for associating specific safety systems with specific applications? Given that no biosafety system can completely eliminate the risk of HGT, how safe is safe enough? Who should decide when the benefits of GEOs outweigh the risks, and what information do they need? We have collected and indexed existing iGEM biosafety projects. We have engaged expert and public opinion to develop a new proposal for qualitative and quantitatve documentation of BioBrick safety devices.<br />
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<br><br> Biosafety is an exciting design challenge, an essential enabling technology for synthetic biology, and a fundamental ethical obligation of all bioengineers. We expect that modular containment systems like bWARE will be standard ware in the next generation of iGEM projects.<br />
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<h3>Go to</h3><br><br />
<center><a href=https://2012.igem.org/Team:Paris_Bettencourt/Achievements> <img src=https://static.igem.org/mediawiki/2012/0/0c/ParisB_achievement_icon.png width=150></a></center><br />
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<center><a href=https://2012.igem.org/Team:Paris_Bettencourt/Overview> <img src=https://static.igem.org/mediawiki/2012/e/e8/ParisB_project_icon.png width=150></a></center><br />
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<center><a href=https://2012.igem.org/Team:Paris_Bettencourt/Human_Practice/Frontpage> <img src=https://static.igem.org/mediawiki/2012/0/05/ParisB_human_icon.png width=150></a></center><br />
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<li><span class="date" style="background:rgb(121,173,89);">9/7/12</span> <a href="/Judging/Track_Selection">Track selection due </a></li><br />
<li><span class="date" style="background:rgb(121,173,89);">9/7/12</span> <a href="/Jamboree/Project_abstract">Project abstracts due </a></li><br />
<li class="last"><span class="date" style="background:rgb(121,173,89);">9/7/12</span> <a href="/Jamboree/Team_Roster">Team rosters due</a></li><br />
<li class="last"><span class="date" style="background:rgb(121,173,89);">9/7/12</span> <a href="/Safety">Safety questions due </a></li><br />
<li class="last"><span class="date">9/26/12</span>Project and part documentation due, including documentation for all medal criteria </li><br />
<li class="last"><span class="date" style="background:rgb(121,173,89);">9/26/12</span>BioBrick Part DNA due to the Registry </li><br />
<li class="last"><span class="date">9/26/12</span> <a href="https://igem.org/2012_Judging_Form?id=914">Judging form due </a></li><br />
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<li class="last"><span class="date">9/26/12</span>Wiki FREEZE at 11:59pm, EDT </li><br />
<li class="last"><span class="date">10/5-7/12</span>iGEM 2012 Regional Jamborees</li><br />
<li class="last"><span class="date">11/2-5/12</span>iGEM 2012 World Championship Jamboree, MIT</li><br />
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<h3>Quick Link</h3><br />
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<li class="first"><a href="https://2012.igem.org">iGEM 2012</a></li><br />
<li class="first"><a href="http://partsregistry.org/Main_Page">Parts Registry</a></li><br />
<li class="first"><a href="http://www.cri-paris.org/en/cri/">The Center for Research and Interdisciplinarity</a></li><br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/ContactTeam:Paris Bettencourt/Contact2012-09-26T21:18:11Z<p>Aleksandra: </p>
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<div id="grouptitle">Contact Us</div><br />
Here is our email address where you can contact us:<br />
team2012@igem-paris.org<br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandrahttp://2012.igem.org/Team:Paris_Bettencourt/AttributionsTeam:Paris Bettencourt/Attributions2012-09-26T21:16:25Z<p>Aleksandra: /* Responsibilities in the Team */</p>
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<div id="grouptitle">Attributions</div><br />
==Responsibilities in the Team==<br />
<br />
All of the designs, constructs (unless stated otherwise) and experiments presented in this wiki were performed by the members of the 2012 Paris Bettencourt team. The advisors and instructors were providing feedback and advice, when needed. None of the subjects of this project are being studied or developed in the hosting lab.<br />
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All members of the team were following the development of the project as a whole. However, in order to be able to work on several modules simultaneously, we formed teams of 1 or 2 that were mainly responsible for each part. <br />
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<div><b>Delay system:</b> Ernest Mordret<br />
<div><b>Semantic containment:</b> Jean Cury<br />
<div><b>Restriction enzyme system:</b> Denis Samuylov and Claire Mayer<br />
<div><b>MAGE:</b> Guillaume Villain and Zoran Marinkovic<br />
<div><b>Suicide system:</b> Julianne Rieders and Aishah Prastowo<br />
<div><b>Encapsulation:</b> Dylan Iverson<br />
<div><b>Synthetic Import Domain:</b> Zoran Marinkovic and Guillaume Villain<br />
<div><b>Human practice:</b> Claire Mayer and Jean Cury<br />
<div><b>Wiki layout:</b> Jean Cury<br />
<div><b>Stop motion:</b> Dylan Iverson, Jean Cury and Julianne Rieders<br />
<div><b>Bonus:</b> Aishah Prastowo and Julianne Rieders<br />
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==External help==<br />
We are extremely thankful to all the following labs, iGEM teams, researchers we met, and other people, for their help :<br />
* Sara Aguiton for her precious advice on the human practice report.<br />
* Professor Mamzer-Bruneel, Professor Gouyon, Professor Morange, Professor Ricroch for the interviews.<br />
* Professor Yokobayashi, for the sRNA repression plasmidic system<br />
* Osnat Gillor, for the Colicin E2 strains<br />
* Miklos de Zamaroczy, for the Colicin D strain<br />
* Bethan and Philip from the UCl iGEM team for participating in our debate and for their discusion and feedback.<br />
* Grégory Hansen and Jean-Baptise Lugagne from the grenoble iGEM team for participating in our debate, and for their feedback.<br />
* Dr. Rosenberg, for the SMR6316 strain with encoded I-SceI endonuclease.<br />
* Dr. Josef Altenbuchner, for the plasmid pJOE3075 with encoded Rhamnose promoter.<br />
* Esengul Yildirim and TUDelft iGEM 2012 team for sending us two biobricks: [http://partsregistry.org/Part:BBa_K175027 BBa_K175027] and [http://partsregistry.org/Part:BBa_K175041 BBa_K175041].<br />
* Theo Sanderson of Cambridge iGEM 2010 team for advice on use of the lux brick.<br />
* Myelin Haoqian Zhang for help with mercury project.<br />
* Bristol 2010 iGEM team for their wonderful Nitrate reporter.<br />
<!--Each team must clearly attribute work done by the team on this page. They must distinguish work done by the team from work done by others, including the host labs, advisors, instructors, graduate students, and postgraduate masters students.--><br />
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==Cooperation==<br />
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* iGEM Grenoble team: <br />
*:We helped them improve their safety sheet. <br />
*:2 team members came to our debate, one student was part of the adjudication pannel, they gave us detaile feedback at the end.<br />
* iGEM UCL team: <br />
*:2 team members came to Paris. We helped them set up meetings with some researchers in Paris from La Paillasse and Fabelier, and offered them crash. <br />
*:They participated to the debate as part of the adjudication pannel, and gave us detailed feedback and the end. <br />
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{{:Team:Paris_Bettencourt/footer}}</div>Aleksandra