Team:Pitt/Skin Probiotic/Cathelicidin/Intro

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<h2>Protocol Design Intro</h2>
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<h2>Cathelicidin Introduction</h2>
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<p>In order to insert genes into P. acnes, we need to establish a proper transformation protocol. However, P. acnes are notoriously hard bacteria to transform because of the strength of the cell wall, and because of the wide range of restriction enzymes present in the cytoplasm. Our general approach is to weaken the cell wall of P. acnes during transformation, increasing the amount of DNA that will enter the cell, and to inhibit the restriction enzymes of P. acnes, increasing the amount of DNA that will stay in the cell. In addition, the plasmid used for our experiments, pBRESP36a, originates from a unique bacterium closely related to P. acnes, whereas most of the iGEM Bio-Bricks originate from E. coli.</p>
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<center><img src = "https://static.igem.org/mediawiki/2014/9/91/Pitt_cath_intro.png" style = "width:500px;"></center>
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<p>The objective of this part of the project was to engineer P. Acnes already on our skin to produce an anti-microbial peptide, which would reduce inflammation by inhibiting local bacteria growth. We chose Cathelicidin as our anti-microbial peptide because it is a natural antibiotic produced by the immune cells of many mammals, including humans.</p>
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<p>We are using a 2-level, partial factor, statistical design of experiments (DOX) to optimize the transformation efficiency of P. acnes through electroporation. Through DOX, we can maximize the number of variables tested, while minimizing the number of trials run. For this project, we decided to evaluate 8 different parameters: The strain of P. acnes, temperature in which P. acnes is grown, amount of glycine added, amount of lysozyme, amount of the plasmid DNA (pBRESP36A), presence of restriction enzyme inhibitor (TypeOne), strength of electric field, and the incubation temperature for recovery. Optimizing the transformation protocol of P. acnes greatly increases the ease with which researchers can study P. acnes by transforming P. acnes to perform other functions. Furthermore, these novel functions can also be used to turn P. acnes into a skin probiotic, by secreting compounds beneficent for the skin.</p>
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<p>A key factor of this construct was making sure that the cathelicidin production was not constitutive, leading to the death of all bacteria and destruction of our skin’s natural biome. Therefore, we decided to combine the cathelicidin production with the YF1/FixJ and FixK2 blue light induction system. This allows regulation of cathelicidin production by blue light, giving the means to suppress the P. Acnes when, and only when, it becomes elevated.</p>
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<p>When designing this system, four main parts were going to be objectives:</p>
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<ol><li>hsp60 (no RBS) – mRFP1<br>
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<center><img src = "https://static.igem.org/mediawiki/2014/6/69/Pitt_cath_intro2.png"></center><br>
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This objective of this part was to test the novel promoter hsp60 with the fluorescence of mRFP1.</li>
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<li>Blue Promoter (FixK2) – (RBS) mRFP1 – hsp60 (no RBS) – Blue Sensor (YF1/FixJ)<br>
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<center><img src = "https://static.igem.org/mediawiki/2014/7/73/Pitt_cath_intro3.png"></center><br>
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The objective of this part was to test the blue light system with hsp60 and mRFP1. Hsp60 would cause constitutive production of the Blue Light Sensor (YF1/FixJ), which in turn, would cause the Blue Light Promoter to cause production of mRFP1 when exposed to blue light. </li>
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<li>Blue Promoter – (RBS) mRFP1 – Cathelicidin – Terminator – hsp60 (no RBS) – Blue Sensor<br>
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<center><img src = "https://static.igem.org/mediawiki/2014/c/ce/Pitt_cath_intro4.png"></center><br>
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The objective of this part was to test the entire system. Hsp60 would cause constitutive production of the Blue Light Sensor. When exposed to blue light, the Blue Light Promoter would activate and cause production of mRFP1 and Cathelicidin. It could therefore be expected that if the cells fluoresced red, that Cathelicidin was being produced.</li>
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<li>Blue Promoter – RBS – Cathelicidin – Terminator – hsp60 (no RBS) – Blue Sensor<br>
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<center><img src = "https://static.igem.org/mediawiki/2014/0/08/Pitt_cath_intro5.png"></center><br>
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This part would be the final product. If the three previous tests were successful, then this part could be expected to produce Cathelicidin when exposed to blue light. This function, without the mRFP1, would be the optimal product to apply to P. Acnes on peoples' skin.</li>
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</ol>
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<center><img src = "https://static.igem.org/mediawiki/2014/thumb/4/4e/Pitt_cath_intro6.png/800px-Pitt_cath_intro6.png"></center>
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<a href = "https://2014.igem.org/Team:Pitt/HSP60_Promoter/Results">
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<a href = "https://2014.igem.org/Team:Pitt/Skin_Probiotic/Cathelicidin/Methods">
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Latest revision as of 19:57, 17 October 2014

Cathelicidin Introduction

The objective of this part of the project was to engineer P. Acnes already on our skin to produce an anti-microbial peptide, which would reduce inflammation by inhibiting local bacteria growth. We chose Cathelicidin as our anti-microbial peptide because it is a natural antibiotic produced by the immune cells of many mammals, including humans.

A key factor of this construct was making sure that the cathelicidin production was not constitutive, leading to the death of all bacteria and destruction of our skin’s natural biome. Therefore, we decided to combine the cathelicidin production with the YF1/FixJ and FixK2 blue light induction system. This allows regulation of cathelicidin production by blue light, giving the means to suppress the P. Acnes when, and only when, it becomes elevated.

When designing this system, four main parts were going to be objectives:

  1. hsp60 (no RBS) – mRFP1

    This objective of this part was to test the novel promoter hsp60 with the fluorescence of mRFP1.
  2. Blue Promoter (FixK2) – (RBS) mRFP1 – hsp60 (no RBS) – Blue Sensor (YF1/FixJ)

    The objective of this part was to test the blue light system with hsp60 and mRFP1. Hsp60 would cause constitutive production of the Blue Light Sensor (YF1/FixJ), which in turn, would cause the Blue Light Promoter to cause production of mRFP1 when exposed to blue light.
  3. Blue Promoter – (RBS) mRFP1 – Cathelicidin – Terminator – hsp60 (no RBS) – Blue Sensor

    The objective of this part was to test the entire system. Hsp60 would cause constitutive production of the Blue Light Sensor. When exposed to blue light, the Blue Light Promoter would activate and cause production of mRFP1 and Cathelicidin. It could therefore be expected that if the cells fluoresced red, that Cathelicidin was being produced.
  4. Blue Promoter – RBS – Cathelicidin – Terminator – hsp60 (no RBS) – Blue Sensor

    This part would be the final product. If the three previous tests were successful, then this part could be expected to produce Cathelicidin when exposed to blue light. This function, without the mRFP1, would be the optimal product to apply to P. Acnes on peoples' skin.


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