Difference between revisions of "Team:Vilnius-Lithuania/Design"

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<h2>Design</h2>
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By talking about your design work on this page, there is one medal criterion that you can attempt to meet, and one award that you can apply for. If your team is going for a gold medal by building a functional prototype, you should tell us what you did on this page. If you are going for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Applied Design award</a>, you should also complete this page and tell us what you did.
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        <h3 class="text-heading">Project design</h3>
  
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        <h3 style="border-left: 5px solid rgb(236,151,31); padding-left: 5px; border-bottom: none">Regulatory unit cloning</h3>
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<h4>Note</h4>
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          <p class="text-justify">Firstly, we synthesized a pair of oligonucleotide primers, which code for pLux/cI (I1051) promoter sequence. We amplified this promoter sequence with the help of polymerase chain reaction (PCR). These promoters were further cloned into LuxI (K805016) and LuxR (I0462) genes.</p>
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<p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best Applied Design award</a> and/or the <a href="https://2015.igem.org/Judging/Awards#Medals">functional prototype gold medal criterion</a>, you must fill out this page.</p>
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<p>This is a prize for the team that has developed a synthetic biology product to solve a real world problem in the most elegant way. The students will have considered how well the product addresses the problem versus other potential solutions, how the product integrates or disrupts other products and processes, and how its lifecycle can more broadly impact our lives and environments in positive and negative ways.</p>
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          <p class="text-justify">We performed one more cloning: pLac operon (R0010) was cloned nearby cI repressor (P0151), which was further cloned into LuxI construct. Finally, we cloned cI+LuxI construct into a plasmid with a LuxR gene. This is our regulatory unit, which, in fact, has all the necessary elements to function and control Coliclock system.</p>
  
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        <h3 style="border-left: 5px solid rgb(236,151,31); padding-left: 5px; border-bottom: none">Functional unit cloning</h3>
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If you are working on art and design as your main project, please join the art and design track. If you are integrating art and design into the core of your main project, please apply for the award by completing this page.
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        <p class="text-justify">Firstly, we synthesized pLuxI/cI promoter sequences, togerther with different RBS sequences: strong (Bba_B0030), medium (Bba_0032) and weak (Bba_0030). Overlapping oligonucleotides were synthesized to the full constructs during PCR. Functional unit, as mentioned before, consists of three main constructs: Cascade complex, cas3 protein and homogenic CRISPR region. CRISPR regions were synthesized by gene synthesis, while Cascade and cas3 proteins were cloned in our laboratory earlier.</p>
  
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      <p class="text-justify">In wild type CRISPR-Cas system proteins we found restriction sites, that are recognized by the restriction enzymes used in Standart Assembly. We aimed to mutate these restriction sites. Cas3 gene had 3 different restriction sites of our interest – EcoRI, XbaI and PstI. Cascade complex had only one restriction site – EcoRI. We performed Quick change gene mutagenesis with the Invitrogen Multi site mutagenesis Plus kit. We changed one nucleotide in that way, that the amino acid, which is coded in the triplet, would not change, however, the restriction site would be damaged. For that reason restrictase would not be able to recognize the site and the middle of the gene would not be cut, when cloning via standart assembly. We sequenced the genes after mutagenesis to make sure that it was successful.</p>
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        <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none">Diagram 1. Cascade after mutagenesis. There is no more EcoRI restriction site, so restriction enzyme should not have any effect on Cascade protein.</p>
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      <p class="text-justify">After that, our team performed PCR reactions, during which we added prefix and suffix sequences to our genes. We also cloned pLux/cI promoters (along with different RBS sequences) into these genes. All in all, we got CRISPR-Cas system genes, which have different expression pattern. By regulating the expression of functional unit we will control the lifetime of Coliclock.</p>
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        <h3 style="border-left: 5px solid rgb(236,151,31); padding-left: 5px; border-bottom: none">References</h3>
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<li>Gasiunas G, Sinkunas T, Siksnys V (2014) Molecular mechanisms of CRISPR-mediated microbial immunity. Cellular and molecular life sciences: CMLS 71: 449-465.</li>
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<li>Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annual review of biochemistry 82: 237-266.</li>
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<li>Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011). Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. The EMBO journal 30: 1335-42.</li>
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<li>Brian J. Caliando, Christopher A. Voigt. Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nature Communications 6, Article number: 6989</li>
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{{Team:Vilnius-Lithuania/Vilnius15_Footer}}

Latest revision as of 17:38, 18 September 2015

Project design

Regulatory unit cloning

Firstly, we synthesized a pair of oligonucleotide primers, which code for pLux/cI (I1051) promoter sequence. We amplified this promoter sequence with the help of polymerase chain reaction (PCR). These promoters were further cloned into LuxI (K805016) and LuxR (I0462) genes.

We performed one more cloning: pLac operon (R0010) was cloned nearby cI repressor (P0151), which was further cloned into LuxI construct. Finally, we cloned cI+LuxI construct into a plasmid with a LuxR gene. This is our regulatory unit, which, in fact, has all the necessary elements to function and control Coliclock system.

Functional unit cloning

Firstly, we synthesized pLuxI/cI promoter sequences, togerther with different RBS sequences: strong (Bba_B0030), medium (Bba_0032) and weak (Bba_0030). Overlapping oligonucleotides were synthesized to the full constructs during PCR. Functional unit, as mentioned before, consists of three main constructs: Cascade complex, cas3 protein and homogenic CRISPR region. CRISPR regions were synthesized by gene synthesis, while Cascade and cas3 proteins were cloned in our laboratory earlier.

In wild type CRISPR-Cas system proteins we found restriction sites, that are recognized by the restriction enzymes used in Standart Assembly. We aimed to mutate these restriction sites. Cas3 gene had 3 different restriction sites of our interest – EcoRI, XbaI and PstI. Cascade complex had only one restriction site – EcoRI. We performed Quick change gene mutagenesis with the Invitrogen Multi site mutagenesis Plus kit. We changed one nucleotide in that way, that the amino acid, which is coded in the triplet, would not change, however, the restriction site would be damaged. For that reason restrictase would not be able to recognize the site and the middle of the gene would not be cut, when cloning via standart assembly. We sequenced the genes after mutagenesis to make sure that it was successful.

Diagram 1. Cascade after mutagenesis. There is no more EcoRI restriction site, so restriction enzyme should not have any effect on Cascade protein.

After that, our team performed PCR reactions, during which we added prefix and suffix sequences to our genes. We also cloned pLux/cI promoters (along with different RBS sequences) into these genes. All in all, we got CRISPR-Cas system genes, which have different expression pattern. By regulating the expression of functional unit we will control the lifetime of Coliclock.

References

  • Gasiunas G, Sinkunas T, Siksnys V (2014) Molecular mechanisms of CRISPR-mediated microbial immunity. Cellular and molecular life sciences: CMLS 71: 449-465.
  • Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annual review of biochemistry 82: 237-266.
  • Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011). Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. The EMBO journal 30: 1335-42.
  • Brian J. Caliando, Christopher A. Voigt. Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nature Communications 6, Article number: 6989