Difference between revisions of "Team:BioIQS-Barcelona/Basic Part"

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                    <div class="header-content header-xl">
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                        <h1 class="mb-5">Wet Lab | Personalization (PCRs)</h1>
 +
                        <a href="https://2018.igem.org/Team:BioIQS-Barcelona/Basic_Part#pcr-person" class="btn btn-outline btn-xl js-scroll-trigger">Have a look!</a>
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                    <h2 class="section-heading orange">Personalization (PCRs)</h2>
 +
                    <div class="col-md-12">
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                        <div class="row justify-content-center block-desc-b">
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                            <div class="col-md-8">
 +
                                <p class="book orange"><i>If you ever considered isolating your HLA-DQ genes from your genomic DNA, here is how.</i></p>
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                    <div class="col-md-12 mx-auto block-sept">
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                        <a class="js-scroll-trigger" href="https://2018.igem.org/Team:BioIQS-Barcelona/Basic_Part#pcr-person#cl-description"><span class="arrow down"></span></a>
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                </div>
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            </div>
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    </section>
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                        <div class="col-md-5">
 +
                            <h3 class="orange-intense">The strategy</h3>
 +
                            <p class="book orange">One of the objectives of our project is to obtain the HLA-DQ protein from scratch. Based on former studies, only the <b>exons 2 and 3 form each chain (&alpha; and &beta;)</b> codify for the extracellular domain of the HLA-DQ that <b>interacts with gluten epitopes.</b></p>
 +
                            <p class="book orange">With this in mind, we designed a robust model for the extraction of the &alpha; and &beta; chains of the HLA-DQ from the genomic DNA of a celiac patient. <b>A set of primers were designed to conduct 3 different PCRS (including 10 reactions)</b> to obtain the &alpha; and &beta; chains flanked with restriction sites for further cloning.</p>
 +
 +
                        </div>
 +
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 +
                            <div class="col-md-12 mx-auto image">
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                                <img class="img-f" src="https://static.igem.org/mediawiki/2018/b/ba/T--BioIQS-Barcelona--2018_aa_estrategia.png">
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                            </div>
 +
                        </div>
 +
                        <div class="col-md-6 right block-desc-t">
 +
                            <h3 class="orange-intense">PCR 1</h3>
 +
                            <p class="book orange">In the PCR1, amplification of the exons 2 and 3 is carried out by using primers P1-P2/P1’-P2’ and P3-P4/P3’-P4’ respectively in <b>4 separated reactions, called PCR_E2A/PCR_E2B and PCR_E3A/PCR_E3B</b>. The product size correspond to the ones expected, <b>253bp</b> and <b>281bp</b> for the exons that codify for the &alpha; chain, and <b>391-328bp</b> for the exons that codify for the &beta; chain.
 +
                        </p></div>
 +
                        <div class="col-md-6 left block-desc-t">
 +
                            <div class="col-md-12 mx-auto image">
 +
                                <img src="https://static.igem.org/mediawiki/2018/f/fc/T--BioIQS-Barcelona--2018_pcr1.png">
 +
                            </div>
 +
                        </div>
 +
                        <div class="col-md-6 right">
 +
                            <h3 class="orange-intense">PCR 2</h3>
 +
                            <p class="book orange">The products of the beforementioned reactions are to be the template of the following PCR2. In this step, the restriction sites NdeI at 5' (in the case of exon 2) and SalI at 3' (for exon 3) are introduced for <b>further cloning in the vector pET22b(+)</b>. Besides, a complementary region between exon 2 (3' end) and 3 (5' end) for further assembling is also introduced. To accomplish this, 4 separated reactions called PCR_HE2A/PCR_HE2B and PCR_HE3A/PCR_HE3B are carried out using primers P1*-P2*/P1’-P2’ and P3*-P4*/P3’-P4’ respectively. The resulting products with the restriction sites and overhangs should be of <b>264</b> and <b>292 bp</b> for the &alpha; chain and <b>289</b> and <b>298 bp</b> for the &beta; chain.
 +
                        </p></div>
 +
                        <div class="col-md-6 left center">
 +
                            <div class="col-md-12 mx-auto image">
 +
                                <img src="https://static.igem.org/mediawiki/2018/d/dd/T--BioIQS-Barcelona--2018_pcr2.png">
 +
                            </div>
 +
                        </div>
 +
                        <div class="col-md-6 right">
 +
                            <h3 class="orange-intense">PCR 3</h3>
 +
                            <p class="book orange">Finally, in the PCR3, assembling of the exons 2 and 3 is done in one single reaction (Final PCR&alpha;/Final PCR&beta;), <b>using a mix of the products from PCR_HE2A and PCR_HE3A</b> (to obtain the &alpha; chain) and <b>PCR_HE2B and PCR_H3B</b> (to obtain the &beta; chain) and the flanking primers P1*-P4*/ P1’-P4’. The expected product size are <b>533 bp</b> (for the &alpha; chain) and <b>566 bp</b> for the &beta; chain).
 +
                        </p></div>
 +
                        <div class="col-md-6 left">
 +
                            <div class="col-md-12 mx-auto image">
 +
                                <img class="img-f" src="https://static.igem.org/mediawiki/2018/3/3f/T--BioIQS-Barcelona--2018_pcr3def.png">
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
        </div>
 +
 +
    </section>
 +
 +
    <section class="project text-center" id="pcr-person">
 +
        <div class="container">
 +
            <div class="row">
 +
                <div class="col-md-9 mx-auto">
 +
                    <h2 class="section-heading orange">Collaborations</h2>
 +
                    <div class="col-md-12">
 +
                        <div class="row justify-content-center block-desc-b">
 +
 +
                            <div class="col-md-8">
 +
                                <p class="book orange"><i>We considered testing the HLA gene extraction protocol that we designed during this summer using a different DNA sample.</i></p>
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                    <div class="col-md-12 mx-auto block-sept">
 +
                        <a class="js-scroll-trigger" href="https://2018.igem.org/Team:BioIQS-Barcelona/Basic_Part#"><span class="arrow down"></span></a>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
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 +
    <section class="st-description block-desc" id="cl-description">
 +
        <div class="container">
 +
            <div class="row">
 +
                <div class="col-md-12 mx-auto">
 +
                    <div class="row block-desc-b">
 +
                        <div class="col-md-5">
 +
                            <h3 class="orange-intense">UPF CRG Barcelona Results</h3>
 +
                            <p class="book orange">The UPF iGEM conducted 3 PCRs for the obtention of the &beta; chain of the HLA-DQ. They used a different DNA sample for demonstrating that the designed protocol was robust. We provided them with all the reagents necessaries to do it, including primers and the iPRoof HF. The primers designed efficiently hybridized with the exons 2 and 3 of the &beta; chain.</p>
 +
                            <p class="book orange">The resulting product flanked with the restriction sites is ready to be cloned in a pET22b vector!</p>
 +
                            <p class="book orange">Congratulations UPF CRG Barcelona! </p>
 +
 +
                        </div>
 +
                        <div class="col-md-7 center">
 +
                            <div class="col-md-12 mx-auto image">
 +
                                <img class="img-f" src="https://static.igem.org/mediawiki/2018/f/f4/T--BioIQS-Barcelona--2018_pcr_collaboration.png">
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                    <p class="orange">Note that P1,P2,P3,P4,P1*,P2*,P3* and P4* refer to the primers used to obtain the &alpha; chain, whereas P1',P2',P3’,P4’,P1’,P2’,P3’* and P4’* were used to obtain the &beta; chain.</p>
 +
                    <div class="col-md-4 button">
 +
                        <a class="text-transform" href="https://2018.igem.org/Team:BioIQS-Barcelona/primer_list.pdf" target="_blank">View &amp; download PDF <i class="fas fa-file-download"></i></a>
 +
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<p> A basic part is a functional unit of DNA that cannot be subdivided into smaller component parts. BBa_R0051 is an example of a basic part, a promoter regulated by lambda cl. </p>
 
  
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<p> Most genetically-encoded functions have not yet been converted to BioBrick parts. Thus, there are many opportunities to find new, cool, and important genetically encoded functions, and refine and convert the DNA encoding these functions into BioBrick standard biological parts. </p>
 
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Revision as of 03:49, 18 October 2018

BIO IQS

Wet Lab | Personalization (PCRs)

Have a look!

Personalization (PCRs)

If you ever considered isolating your HLA-DQ genes from your genomic DNA, here is how.

The strategy

One of the objectives of our project is to obtain the HLA-DQ protein from scratch. Based on former studies, only the exons 2 and 3 form each chain (α and β) codify for the extracellular domain of the HLA-DQ that interacts with gluten epitopes.

With this in mind, we designed a robust model for the extraction of the α and β chains of the HLA-DQ from the genomic DNA of a celiac patient. A set of primers were designed to conduct 3 different PCRS (including 10 reactions) to obtain the α and β chains flanked with restriction sites for further cloning.

PCR 1

In the PCR1, amplification of the exons 2 and 3 is carried out by using primers P1-P2/P1’-P2’ and P3-P4/P3’-P4’ respectively in 4 separated reactions, called PCR_E2A/PCR_E2B and PCR_E3A/PCR_E3B. The product size correspond to the ones expected, 253bp and 281bp for the exons that codify for the α chain, and 391-328bp for the exons that codify for the β chain.

PCR 2

The products of the beforementioned reactions are to be the template of the following PCR2. In this step, the restriction sites NdeI at 5' (in the case of exon 2) and SalI at 3' (for exon 3) are introduced for further cloning in the vector pET22b(+). Besides, a complementary region between exon 2 (3' end) and 3 (5' end) for further assembling is also introduced. To accomplish this, 4 separated reactions called PCR_HE2A/PCR_HE2B and PCR_HE3A/PCR_HE3B are carried out using primers P1*-P2*/P1’-P2’ and P3*-P4*/P3’-P4’ respectively. The resulting products with the restriction sites and overhangs should be of 264 and 292 bp for the α chain and 289 and 298 bp for the β chain.

PCR 3

Finally, in the PCR3, assembling of the exons 2 and 3 is done in one single reaction (Final PCRα/Final PCRβ), using a mix of the products from PCR_HE2A and PCR_HE3A (to obtain the α chain) and PCR_HE2B and PCR_H3B (to obtain the β chain) and the flanking primers P1*-P4*/ P1’-P4’. The expected product size are 533 bp (for the α chain) and 566 bp for the β chain).

Collaborations

We considered testing the HLA gene extraction protocol that we designed during this summer using a different DNA sample.

UPF CRG Barcelona Results

The UPF iGEM conducted 3 PCRs for the obtention of the β chain of the HLA-DQ. They used a different DNA sample for demonstrating that the designed protocol was robust. We provided them with all the reagents necessaries to do it, including primers and the iPRoof HF. The primers designed efficiently hybridized with the exons 2 and 3 of the β chain.

The resulting product flanked with the restriction sites is ready to be cloned in a pET22b vector!

Congratulations UPF CRG Barcelona!

Note that P1,P2,P3,P4,P1*,P2*,P3* and P4* refer to the primers used to obtain the α chain, whereas P1',P2',P3’,P4’,P1’,P2’,P3’* and P4’* were used to obtain the β chain.

View & download PDF