Team:KULeuven/Details

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KULeuven iGEM 2011

Extended project description

still under construction: Clarifying pictures will be added soon, all biobricks will be clickable and lead towards the biobrick register

This summer, we are engineering the bacterium E.D. Frosti that induces ice crystallization, using the ice-nucleating protein ( INP), or will inhibit ice crystal formation, using the anti-freeze protein ( AFP), depending on the given stimulus. These proteins will be extracellularly anchored at E.D. Frosti’s cell membrane. Furthermore, it is essential to create a dual-inhibition system, so that INP and AFP can never be expressed at the same time. To verify the function of this system, we coupled the production of a specific color depending on the stimulus given to E.D. Frosti. Finally, as a safety mechanism, we installed a suicide mechanism in E.D. Frosti, whose activity is mediated by an “AND”-gate system: the cell death mechanism is only activated when one of the two stimuli is given, AND a sudden decrease in temperature (a cold-shock) occurs.
To realize our E.D. Frosti project, we had to create several mechanisms which will be outlined below.

1. Dual inhibition system

Since E.D. Frosti has all the genetic information to exercise both functions, it is very important to make sure that only one type of proteins will be expressed in one E.D. Frosti cell. A dual inhibition system is a good way to ensure this and is explained in the next figure.
When arabinose is added to the medium (stimulus 1), it will induce transcription from the pBAD promoter ( BBa_I13453 ), resulting in LuxR ( BBa_I0462 ) and LuxI ( BBa_C0261 ) transcripts that are translated into the respective proteins. LuxI is an enzyme that catalyzes the production of N-Acyl homoserine lactones ( AHLs ) from S-adenosyl methionine (SAM ) and acyl-coenzyme A (acyl-CoA). These AHLs, when bound to LuxR, are able to regulate transcription through binding with a luxR binding site located in promoter regions. In our system, the LuxR-AHL complex will perform a dual task.
While it activates the pLux-CI promoter ( BBa_R0065), resulting in the transcriptional activation of OmpA-AFP (NEW BIOBRICK)and MelA ( BBa_K193602 ) (2), it acts as a negative regulator of the pLac-Lux promoter ( BBa_K091100), the transcriptional regulator of stimulus 2, an additional safety mechanism to ensure that no INP can be produced during stimulus 1, even under non-inducing conditions. If we add lactose or IPTG to the medium (stimulus 2), it will activate transcription from the pLac-Lux promoter , resulting in INP (NEW BIOBRICK), CI repressor and CrtEBI ( BBa_K274100 ) transcripts that are translated into their specific proteins. The CI repressor will repress the pLux-CI promoter and, thereby specifically inhibiting the production of AFP, while INP is being expressed.

@@ Line 117: / Line 117: @@
 <b><u><i> still under construction: Clarifying pictures will be added soon, all biobricks will be clickable and lead towards the biobrick register </b></u></i><br><br>
-Our system consists of 4 components which are outlined underneath: a regulation plasmid; an antifreeze component; a freeze component and a cell death mechanism.<br><br>
+This summer, we are engineering the bacterium <i>E.D. Frosti</i> that induces ice crystallization, using the ice-nucleating protein (<a href=”https://2011.igem.org/Team:KULeuven/Inp” target= “blank”> INP</a>), or will inhibit ice crystal formation, using the anti-freeze protein (<a href=”https://2011.igem.org/Team:KULeuven/Afp” target=”blank”> AFP</a>), depending on the given stimulus. These proteins will be extracellularly anchored at <i>E.D. Frosti</i>’s cell membrane. Furthermore, it is essential to create a dual-inhibition system, so that INP and AFP can never be expressed at the same time. To verify the function of this system, we coupled the production of a specific color depending on the stimulus given to <i> E.D. Frosti</i>. Finally, as a safety mechanism, we installed a <a href=”https://2011.igem.org/Team:KULeuven/Input” target=”blank”> suicide mechanism</a > in <i> E.D. Frosti</i>, whose activity is mediated by an “AND”-gate system: the cell death mechanism is only activated when one of the two stimuli is given, AND a sudden decrease in temperature (a cold-shock) occurs. <br>To realize our <i>E.D. Frosti</i> project, we had to create several mechanisms which will be outlined below.<br><br>
+<h2>1. Dual inhibition system</h2>
+Since <i> E.D. Frosti</i> has all the genetic information to exercise both functions, it is very important to make sure that only one type of proteins will be expressed in one <i> E.D. Frosti</i> cell. A dual inhibition system is a good way to ensure this and is explained in the next figure. <br>
-<h2> 1: Regulation plasmid </h2>
+When arabinose is added to the medium (stimulus 1), it will induce transcription from the  <i>pBAD</i> promoter (<a href=”http://partsregistry.org/Part:BBa_I13453” target=”blank”> BBa_I13453 </a>), resulting in  <i>LuxR</i> (<a href=” http://partsregistry.org/Part:BBa_I0462” target=”blank”> BBa_I0462</a> ) and <i>LuxI</i> (<a href=” http://partsregistry.org/Part:BBa_C0261” target=”blank”> BBa_C0261</a> ) transcripts that are translated into the respective proteins. LuxI is an enzyme that catalyzes the production of N-Acyl homoserine lactones (<a href=”http://en.wikipedia.org/wiki/Homoserine_Lactone” target=”blank” > AHLs </a>) from S-adenosyl methionine (SAM )  and acyl-coenzyme A (acyl-CoA).  These AHLs, when bound to LuxR, are able to regulate transcription through binding with a luxR binding site located in promoter regions. In our system, the LuxR-AHL complex will perform a dual task. <br>While it activates the <i>pLux-CI</i> promoter (<a href=” http://partsregistry.org/Part:BBa_R0065” target=”blank”> BBa_R0065</a>), resulting in the transcriptional activation of <i>OmpA-AFP</i> (<b>NEW BIOBRICK</b>)and <i>MelA </i> (<a href=” http://partsregistry.org/Part:BBa_K193602” target=”blank”> BBa_K193602 </a>) (2),  it acts as a negative regulator of the <i>pLac-Lux</i> promoter (<a href=” http://partsregistry.org/Part:BBa_K091100” target=”blank”> BBa_K091100</a>), the transcriptional regulator of stimulus 2, an additional safety mechanism to ensure that no INP can be produced during stimulus 1, even under non-inducing conditions.
-Transcription of this component will activate the AFP and repress the INP formation. Activation proceeds through the luxR protein (Bba_C0062) and repression through the luxI protein (Bba_C0061). LuxR is an activator of the Lux-CI promoter which is used in our antifreeze component. At the other hand luxI is a repressor of the Lac-lux promoter, which is used in our freeze component, so that both genes (<i>luxR</i> and<i>luxI</i>) are under the control of the pBAD promoter (Bba_I3453) which is induced when L-arabinose is present. [A picture of these biobricks will be uploaded]<br><br>
+If we add lactose or IPTG to the medium (stimulus 2), it will activate transcription from  the <i>pLac-Lux</i> promoter , resulting in  <i>INP</i> (<b>NEW BIOBRICK</b>), <i>CI repressor</i> and <i>CrtEBI</i> (<a href=” http://partsregistry.org/Part:BBa_K274100 “ target=”blank”> BBa_K274100 </a>) transcripts that are translated into their specific proteins. The CI repressor will repress the <i>pLux-CI</i> promoter and, thereby specifically inhibiting the production of AFP, while INP is being expressed.
-<h2> 2: Antifreeze</h2>
-The purpose of this component is to synthesize an antifreeze protein after a given stimulus, in this case arabinose. Normally, the antifreeze protein is produced intracellularly to prevent a cell from freezing. We however, try to prevent the environment from freezing and therefore we design a system that couples the antifreeze protein to the extracellular membrane. To check if we really have transcribed the protein we couple melanin production to the promoter as well. Melanin is a black pigment and thus visualizes the antifreeze production.<br><br>
-The most important biobrick in the antifreeze system is the extracellular antifreeze protein. It is a new biobrick we want to synthesize. In order to couple the antifreeze protein to the extracellular membrane, we fuse it to an extracellular anchor (Bba_K103006) and a flexible linker (BBa_K105012 ). Also the antifreeze protein (AFP) itself  is a new biobrick.[A picture of this new biobrick will be added] <br><br>
-The second protein that we will use in the antifreeze system, is the enzyme tyrosinase (EC 1.14.18.1) (BBa_K193600). This enzyme converts tyrosine to dopaquinone. Dopaquinone is an intermediate product of melanin biosynthesis pathway. After production of Dopaquinone, Melanin is generated by non-enzymatical chain reaction, so overexpression of tyrosinase in <i>E.coli</i> causes accumulation of Melanin. (reference: https://2009.igem.org/Team:Tokyo_Tech/DarkercoloredEcoli) The Tokyo tech team of 2009 showed that the color of the strain expressing tyrosinase became 10-fold darker than that of the control.<br><br>
-The promoter we use for both proteins is the plux-CI promotor (BBa_R0065). It is a hybrid promoter responding to cI repressor and LuxR. CI repressor negatively regulates this promoter and LuxR activates its transcription. The effect of cI is dominant over LuxR.<br><br>
-<h2>3: Freeze</h2>
-This component induces water to freeze through the use of Ice Nucleating Proteins (INPs). INPs are already anchored in the outer membrane through its N-terminal and C-terminal region. In order to check if we really have transcribed the protein, we couple the production of luminescent pigment to the promoter as well. This bright pigment, lets us visualize the freezing system.<br><br>
-Two other biobricks used in Freeze component are crtB coding sequence encoding phytoene synthase and crtI coding sequence encoding phytoene dehydrogenase.  The first one is the coding sequence of crtI from <i>Pantoea ananatis</i> (formerly <i>Erwinia uredovora</i>) (Accession number D90087). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene (Misawa, et al., 1990).<br><br>
-The second one is the coding sequence of crtI from <i>Pantoea ananatis</i> (formerly <i>Erwinia uredovora</i>) (Accession number D90087). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene (Misawa, et al., 1990).  <br><br>
-The promoter we use is the pLac-lux hybrid promoter, BBa_K091100. This promoter is repressed by the lacI repressor and induced by IPTG. It is repressed by the luxR repressor bound to the corepressor CO6HSL. It contains a lux box and a lacI binding site. Besides, The cI regulated promoter is based on the pR promoter from bacteriophage <i>lambda</i>. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. <br><br>
-<h2>4: cell death mechanism</h2>
-The cell death mechanism we use will be induced when stimulus 1 (L-arabinose) or stimulus 2 (lactose) is given and at the same time a cold temperature is sensed by the bacterium. <br><br>
-For this we created the following procedure: we grow the bacteria at the optimal temperature of 37°C. Then we produce the protein we wish by applying the correct stimulus (e.g., L-arabinose to produce AFP). After synthesizing the desired amount of protein, we lower the temperature to induce the cell death mechanism.<br><br>
-The most essential protein for inducing cell death is the colicin activity gene, CeaB (Bba_K131009). CeaB is part of the Colicin E2 operon. The operon comprises the colicin activity gene, ceaB, the colicin immunity gene, ceiB, and the lysis gene, celB, which is essential for colicin release from producing cells. We only use CeaB , because we only have to kill the inner system of the cell and not the membrane with its useful proteins (AFP or INP). Via this way we also try to prevent horizontal exchange of DNA between <i>E.D. Frosti</i> and other bacteria.<br><br>
-To obtain the desired regulation of the cell death mechanism, a key-lock mechanism is implemented. A ribokey is under regulation of a cold sensing promoter, either CspA promoter (Bba_K328001) or HybB promoter (Bba_K410000). This ribokey is the key to unlock the ribolock, which happens when cold conditions are applied. The ribolock prevents the CeaB protein to be transcribed under normal conditions (37°C). Only cold temperatures are not sufficient to induce cell death, also a stimulus associated with INP- or AFP generation is needed. This prevents cells from being killed when stored in the fridge before generation of INP or AFP. To become dependent on a second stimulus we put the same promoters as for generation of INP or AFP in front of the ribolock. These promoters are the Lux-CI promoter (Bba_R0065) (the same promoter as for generation of AFP) and Lac-Lux promoter (Bba_K091100) (the same promoter as for generation of INP).<br><br>