Difference between revisions of "Team:Austin LASA/Design"

Revision as of 02:38, 18 October 2018

const p = t => h('p', null, t);
h(g.Page, {title: 'Design, Development, and Results', prev: 'https://2018.igem.org/Team:Austin_LASA/Description', next: 'https://2018.igem.org/Team:Austin_LASA/Notebook', selector: [2, 1]},
  h(g.Section, {title: 'What will be used to detect HIV?'},
    p('Because our final Cas12a detection assay relies on sequence recognition between the crRNA spacer sequence and a target strand, we only needed to choose specific sequences from HIV’s genome to work with.'),
  ),
  h(g.Section, {title: 'Choosing a Gene'},
    p('In the end, we chose to work with one of HIV’s genes to simplify our work in the lab. Because HIV is a BSL3 organism, we were unable to work with HIV or any HIV-infected human cells in the lab per iGEM’s Biosafety Standards. Instead, we worked with synthetic viral DNA that we ordered as gblocks through IDT.'),
    p('We ultimately focused on Rev, a non-hazardous gene in HIV’s genome which codes for a transportation protein in HIV. The sequence we found for Rev was specifically from HIV1, and as a result, our subsequent work in the lab focused on the detection of HIV-1.'),
    p('Rev served as the viral sequence we would attempt to amplify with loop-mediated isothermal amplification (LAMP) and subsequently detect with our Cas12a assay.')
  ),
  h(g.Section, {title: 'Designing crRNA'},
    p('Once we decided on an HIV gene to detect, we went ahead and designed crRNA sequences for our Cas12a assay. crRNA sequences are the basis of our detection assay. However, Cas12a crRNA design is still a field of much study, and it was very difficult to find specific information on how to design crRNA. Ultimately, the design of our crRNA was broken up into two components: the hairpin sequence and spacer sequence.'),
    p('We decided on two hairpin sequences, one for AsCas12a and one for LbCas12a. Our LbCas12a sequence was taken from the Doudna Lab. Our AsCas12a sequence was taken from the Finkelstein Lab. We would test our AsCas12a crRNA with purified AsCas12a enzyme, and we would later attempt to clone a gene circuit expressing LbCas12a to use with our LbCas12a crRNA in our cellular reagents.'),
    p('We used several criteria when choosing our spacer sequence. The most important criteria for choosing our spacer sequence was that it needed to be around 24bp following Cas12a’s PAM site, TTTN. From there, we distinguished between spacer sequences based on the following:'),
    h('ul', null,
      h('li', null, 'GC content'),
      h('li', null, 'Dimer formation'),
      h('li', null, 'Similarities to Cas9 sgRNA design')
    ),
    p('Ultimately, we designed 7 different crRNA sequences, and ultimately used 4.'),
  ),
  h(g.Section, {title: 'Can we detect our gene? Testing out crRNA: Cas12a Assay with Purified Enzyme'},
    p('Following the design of our crRNA, we conducted a Cas12a assay with purified AsCas12a and several of our crRNAs.'),
    p('(See Experiments page for more information on carrying out our Cas12a assay.)')
  ),
  h(g.Section, {title: 'Can we amplify our gene isothermally to increase sensitivity?
LAMP Primer and Amplicon Design'},
    p('Loop-mediated isothermal amplification (LAMP) is an isothermal amplification method that relies on specific primers to continuously create kinks in a LAMP amplicon that contain repeats of the template DNA.'),
    p('A LAMP reaction requires a primer set of four specific primers. Rather than only designing one primer set, we were advised to initially test a large number of primer sets. Our lab has found that LAMP primers will not amplify well, and it must be experimentally confirmed which LAMP primer sets work best. Additionally, during our primer design phase, we realized we would only be able to design primers for regions of a couple hundred base pairs, as opposed to the full 2.7kb of the Rev gene. As a result, using PrimerDesign, we designed six different LAMP primer sets that amplified regions of Rev that also contained our crRNA sequences.'),
    p('However, LAMP amplicons have a very special structure. The largest bulk of the resultant amplicons in a LAMP reaction have a continuous zig-zag shape with hairpin loops at the corners. Our lab has previously done work with oligonucleotide strand displacement with LAMP amplicons and has found they work best at hairpin loops. In our project, we wanted to see whether having the target sequence in the linear region or in the loop region would affect Cas12a’s ability to recognize the sequence.'),
    p('Therefore, when designing our LAMP primer sets and resultant amplicons, we designed primers such that, for each primer set, there would be a target sequence corresponding to one of our designed crRNAs in the linear region of the amplicon and in the loop region of the amplicon.')
  ),

);

@@ Line 30: / Line 30: @@
      ),
      p('Ultimately, we designed 7 different crRNA sequences, and ultimately used 4.'),
+  ),
+  h(g.Section, {title: 'Can we detect our gene? Testing out crRNA: Cas12a Assay with Purified Enzyme'},
+    p('Following the design of our crRNA, we conducted a Cas12a assay with purified AsCas12a and several of our crRNAs.'),
+    p('(See Experiments page for more information on carrying out our Cas12a assay.)')
+  ),
+  h(g.Section, {title: 'Can we amplify our gene isothermally to increase sensitivity?
+LAMP Primer and Amplicon Design'},
+    p('Loop-mediated isothermal amplification (LAMP) is an isothermal amplification method that relies on specific primers to continuously create kinks in a LAMP amplicon that contain repeats of the template DNA.'),
+    p('A LAMP reaction requires a primer set of four specific primers. Rather than only designing one primer set, we were advised to initially test a large number of primer sets. Our lab has found that LAMP primers will not amplify well, and it must be experimentally confirmed which LAMP primer sets work best. Additionally, during our primer design phase, we realized we would only be able to design primers for regions of a couple hundred base pairs, as opposed to the full 2.7kb of the Rev gene. As a result, using PrimerDesign, we designed six different LAMP primer sets that amplified regions of Rev that also contained our crRNA sequences.'),
+    p('However, LAMP amplicons have a very special structure. The largest bulk of the resultant amplicons in a LAMP reaction have a continuous zig-zag shape with hairpin loops at the corners. Our lab has previously done work with oligonucleotide strand displacement with LAMP amplicons and has found they work best at hairpin loops. In our project, we wanted to see whether having the target sequence in the linear region or in the loop region would affect Cas12a’s ability to recognize the sequence.'),
+    p('Therefore, when designing our LAMP primer sets and resultant amplicons, we designed primers such that, for each primer set, there would be a target sequence corresponding to one of our designed crRNAs in the linear region of the amplicon and in the loop region of the amplicon.')
    ),