Difference between revisions of "Team:IIT Delhi/InterLab"

 
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           </div>   
 
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<div class="bgimg-1">
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   <div class="caption">
 
   <div class="caption">
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     <span class="border" >Text anyone?</span>
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     <span class="border" >Fifth International InterLab Measurement Study</span>
 
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<div style="color: #777;background-color:white;text-align:center;padding:50px 80px;text-align: center;">
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<div style="color: #777;background-color:white;text-align:center;padding:50px 80px;text-align: justify;">
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  <p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814001 = pLTL
 
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    <br>
 
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      </p>
 
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;"> The pLTL (Lac-Tet-Lac) is a hybrid promoter. It is a promoter composed of the operator sequences of pTet and the pLac promoter. There are multiple benefits of using the pLTL promoter.
 
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</p>
 
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      <ul style="font-size:20px;" >
 
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      <li style="font-family: 'Lato', sans-serif;font-weight:400;">The hybrid pLTL shows almost complete repression on being repressed, and on induction (by IPTG and/or aTc), the hybrid pLTL promoter shows a 1.4-2 times higher expression.</li>
 
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      <li style="font-family: 'Lato', sans-serif;font-weight:400;">The hybrid pLTL promoter also permits flexible gene expression because it can be utilized under either or both repression controls (LacI and TetR) simultaneously.</li>
 
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    </ul>
 
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<br>
 
  
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814008 = rrnB T1 Terminator + T7Te Terminator
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<p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;color: black;margin: auto;width: 75%;">
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    <br>
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iGEM teams all over the world in association with The Measurement Committee, through the InterLab study, have been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. In order to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab.<br>
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;"> Our part is double terminator composed of rrnB T1 Terminator(BBa B0010) and T7 Te Terminator(BBa B0012). Both of these are forward terminators that are extensively used in E. coli.
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</p>
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<br>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814009 = mKate2
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;"> Mkate2 - mKate2 is a red fluorescent protein derived from Entacmaea quadricolor. It possesses fluorescence with excitation maxima at 588 nm and emission maxima at 588 and 633 nm, mKate2 is almost 3-fold brighter than mKate. mKate2 can be used in   labelling applications along with blue, cyan, green, yellow, and red fluorescent dyes. Its high pH-stability with pKa=5.4 makes it useful for imaging in acidic organelles, such as late and recycling endosomes and lysosomes.
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</p>
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<br>
 
<br>
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iGEM IIT Delhi once again participated in the iGEM InterLab Measurement Study, in order to tackle the issue of variability in bulk measurements of a population of cells (such as with a plate reader).<br>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814010 = rrnB T1 Terminator
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    <br>
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      </p>
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    <br>
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</div>
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<div class="bgimg-2">
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  <div class="caption">
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    <span class="border" >Text anyone?</span>
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<div style="position:relative;">
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  <div style="color: #777;background-color:white;text-align:center;padding:50px 80px;text-align: justify;">
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814007 = sfGFP_ssrA
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    <br>
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">Our part consists of sfGFP appended with a ssrA(LVA) deg-tag. It has been codon optimised for E. coli.  This part is useful as it has high intensity as compared to other gfp variants as well as faster degradation rates and shorter reporter lifetimes.
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</p>
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<p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">Superfolder GFP is a basic (constitutively fluorescent) green fluorescent protein derived from Aequorea victoria. It has an emission wavelength of 510 nm and excitation wavelength of 485nm. It has a robust folding characteristic. The superfolder mutations also make the folding of GFP tolerant of mutations that would otherwise reduce the folding yield of GFP.
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</p>
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<p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">Our ssrA degtag is the gfp(LVA) that has a single (A → G) point mutation in nucleotide 349, resulting in an Asp117 → Gly117 (D117G) amino acid change. This point mutation does not appear to change the fluorescence spectrum of gfp(LVA). The LVA tag has been reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute. This corresponds to in vivo half-lives of mature Gfp(LVA) of approximately 40 min.
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</p>
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<p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">References</p>
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<ol style="font-size: 20px;">
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<li style="font-family: 'Lato', sans-serif;font-weight:400;">Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nature biotechnology. 2006 Jan;24(1):79.</li>
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<li style="font-family: 'Lato', sans-serif;font-weight:400;">Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240–6 (1998).
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</li>
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  </ol>
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<br>
 
<br>
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The idea of this year's study was to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Usually, this is done by measuring the absorbance of light at 600nm, from which we compute the “optical density (OD)” of the sample as an approximation of the number of cells.<br>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814011 = attP TP901-1
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    <br>
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">AttP TP901-1 - It is the AttP site of TP901-1 integrase (serine based recombinase enzyme). TP901-1 integrase is an enzyme that has been isolated from TP901-1 phage. It is responsible for catalysing site-specific recombination at AttP and AttB sites. The recombination mechanism depends on the orientation of the AttP and AttB sites. These enzymes help the virus integrate its DNA into the bacterial genome. TP901-1 is widely used in the construction of logic gates and modification of DNA sequences.
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</p>
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<br>
 
<br>
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In the InterLab Measurement Study, iGEM IITD performed 2 sets of experiments, as required by the InterLab protocol(we used Synergy H1 Microplate Reader),<br>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814012 = BxbI Integrase + ssrA(LVA) deg tag
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    <br>
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">BxbI Integrase triggers attP × attB recombination. The product of attP × attB recombination is an integrated prophage flanked by two new recombination sites, attL and attR, each containing half sites derived from attP and attB. In the absence of accessory factors the integrases mediate unidirectional recombination between attP and attB with greater than 80% efficiency. In the presence of a phage-encoded accessory protein, the recombination directionality factor (RDF) the attP × attB recombination is inhibited and the attL × attR recombination is stimulated.
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Bxb1 integrase yields approximately two-fold more recombinants  and displays about two fold less damage to the recombination sites than other phage-encoded serine integrases.
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Our BxbI Integrase has a ssrA deg tag attached to it for faster degradation rates.
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</p>
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<br>
 
<br>
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<b style="
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814013 = attB TP901-1
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    font-size: 125%;
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">1. Conversion between absorbance of cells to the absorbance of a known concentration of beads</b>
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<br><br>
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    <br>
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We were provided with a sample containing silica beads that are roughly the same size and shape as a typical <i>E. coli</i> cell, so that it should scatter light in a similar way. Since we knew the concentration of the beads, we were able to convert our lab’s absorbance measurements into a standard “equivalent concentration of beads” measurement.<br>
−
      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">AttB TP901-1 -  It is the AttB site of TP901-1 integrase (serine based recombinase enzyme). TP901-1 integrase is an enzyme that has been isolated from TP901-1 phage. It is responsible for catalysing site-specific recombination at AttP and AttB sites. The recombination mechanism depends on the orientation of the AttP and AttB sites. These enzymes help the virus integrate its DNA into the bacterial genome. TP901-1 is widely used in the construction of logic gates and changing DNA sequence.
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</p>
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<br>
 
<br>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814014 = complement of B0034, RBS on the antisense strand, twin exists
 
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    <br>
 
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      </p>
 
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<br>
 
  
 +
<img src="https://static.igem.org/mediawiki/2018/8/8a/T--IIT_Delhi--InterlabParticle2.png" style="border:3px solid #000000" width="48%">
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<img src="https://static.igem.org/mediawiki/2018/d/dc/T--IIT_Delhi--InterlabFluorescin2.png" style="border:3px solid #000000" width="48%">
  
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  <div style="color: #777;background-color:white;text-align:center;padding:50px 80px;text-align: justify;">
 
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814015 = pLac(lambda) hybrid
 
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    <br>
 
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      </p>
 
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">pLac - Lambda hybrid - is a hybrid promoter consisting of Lac and Lambda operator sites in the core region. This hybrid promoter can be induced in the presence of  IPTG or
 
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</p>
 
 
<br>
 
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<br style="
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    font-size: 125%;
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">
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<b style="
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">2. Counting colony-forming units (CFUs) from the sample.</b>
 +
<br><br>
  
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814017 = attP Bxb1
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We determined how many live cells were in the volume of media that we plated out and obtained a cell concentration for our sample as a whole. We determined the number of CFUs in positive and negative control samples in order to compute a conversion factor from absorbance to CFU.<br>
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    <br>
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">AttP Bxb1 - It is the AttP site of Bxb1 integrase (Serine based recombinase). Bxb1 integrase carries out site-specific recombination by catalysing unidirectional recombination to produce AttL and AttR sites from AttP and AttB. This switching capacity allows them to be used in the design of toggle switches.
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</p>
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<br>
 
<br>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814018 = attB Bxb1
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The data for both the experiments were submitted to iGEM, additionally, you can find our measurements for the Plate Reader here : <a href="https://static.igem.org/mediawiki/2018/e/e1/T--IIT_Delhi--Interlab18.xlsx"><b>IIT Delhi InterLab Plate Reader Measurements</b></a>
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    <br>
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">AttB Bxb1 - It is the AttB site of Bxb1 integrase (Serine based recombinase). Bxb1 integrase carries out site-specific recombination by catalysing unidirectional recombination to produce AttL and AttR sites from AttP and AttB. This switching capacity allows them to be used in the design of logic gates.
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</p>
 
</p>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814019 = P7 Promoter
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">Constitutive P7 promoter - It is the complement of the constitutive P7 promoter so as to initiate transcription in the reverse direction. 
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</p>
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<br>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814021 = BxbI-Xis  + ssrA(LVA) deg tag------------
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      </p>
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">Xis-BxbI_ssrA deg tag - Consists of bxb1 excisionase followed by ssrA degradation tag. Bxb1-Xis  catalyses the conversion of AttL and AttR sites to AttP and AttB sites when expressed with Bxb1 integrase, thereby reverting the recombination caused by integrase. ssrA deg tag degrades the Bxb1-Xis formed as an increase in the amount of excisionase renders the system inefficient. This property allows Bxb1 to be used in the construction of logic gates.
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</p>
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<br>
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<p style="font-size: 30px; font-family: 'Roboto', sans-serif;font-weight:700;">BBa_K2814022 = attB BxbI (reverse orientation)
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      <p style="font-size:20px;font-family: 'Lato', sans-serif;font-weight:400;">AttB-BxbI (Reverse Orientation) - Contains AttB site of Bxb1 integrase in the reverse orientation (Reverse of  BBa_K2814018). Bxb1 integrase carries out site-specific recombination by catalysing unidirectional recombination to produce AttL and AttR sites from AttP and AttB. This switching capacity allows them to be used in the design of logic gates.
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</p>
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         <div  style="background-color: #555;color:black;font-size: 35px;text-align: center;font-family: sans-serif;"><h4 style="color: white">Contact us</h4></div>
 
         <div  style="background-color: #555;color:black;font-size: 35px;text-align: center;font-family: sans-serif;"><h4 style="color: white">Contact us</h4></div>
 
             <div style="text-align: center;margin-bottom: -136px;"><h4 style="color:black;font-size: 50px;"><u class="fx" style="cursor: pointer;text-align: center">Address</u></h4><p style="text-align: center;margin-bottom: 16px">Undergraduate Laboratory<br>
 
             <div style="text-align: center;margin-bottom: -136px;"><h4 style="color:black;font-size: 50px;"><u class="fx" style="cursor: pointer;text-align: center">Address</u></h4><p style="text-align: center;margin-bottom: 16px">Undergraduate Laboratory<br>
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Latest revision as of 01:53, 18 October 2018

iGEM IIT Delhi

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Overview Design
Fifth International InterLab Measurement Study

iGEM teams all over the world in association with The Measurement Committee, through the InterLab study, have been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. In order to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab.

iGEM IIT Delhi once again participated in the iGEM InterLab Measurement Study, in order to tackle the issue of variability in bulk measurements of a population of cells (such as with a plate reader).

The idea of this year's study was to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Usually, this is done by measuring the absorbance of light at 600nm, from which we compute the “optical density (OD)” of the sample as an approximation of the number of cells.

In the InterLab Measurement Study, iGEM IITD performed 2 sets of experiments, as required by the InterLab protocol(we used Synergy H1 Microplate Reader),

1. Conversion between absorbance of cells to the absorbance of a known concentration of beads

We were provided with a sample containing silica beads that are roughly the same size and shape as a typical E. coli cell, so that it should scatter light in a similar way. Since we knew the concentration of the beads, we were able to convert our lab’s absorbance measurements into a standard “equivalent concentration of beads” measurement.



2. Counting colony-forming units (CFUs) from the sample.

We determined how many live cells were in the volume of media that we plated out and obtained a cell concentration for our sample as a whole. We determined the number of CFUs in positive and negative control samples in order to compute a conversion factor from absorbance to CFU.

The data for both the experiments were submitted to iGEM, additionally, you can find our measurements for the Plate Reader here : IIT Delhi InterLab Plate Reader Measurements

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Undergraduate Laboratory
Department of Biotechnology and Biochemical Engineering, IIT Delhi