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

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    <span class="border" >Fifth International InterLab Measurement Study</span>
 
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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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<br><br>With the advent of synthetic biology, a lot of synthetic circuits are designed every now and then. These circuits contain a lot of components working in harmony to regulate the gene expression. In any system, as the number of components which work together increases, the need for those components to be orthogonal also increases. The components, if interfere with the working of each other, may cause the system as a whole to fail. Such systems succumb to the noise and growth-related factors present in the biological environment. Recombinases in a biological system are adept at this job and hence, are a good basis for the formation of large-scale networks. Utilizing serine recombinases in such biological circuits provides a solution to these problems, as they have non-identical recognition sites and a particular recombinase works orthogonally to other recombinases.<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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These allow us to build complex circuits with genetic modification in relatively uncomplicated configurations owing to the small size of attB and attP sites(~50bp). The attP and attB sites (and thus the integrase-att complexes) have imperfect two-fold symmetry. Hence they allow recombination in two different ways, with the sites aligned in ‘parallel’ or ‘antiparallel’ configuration. Thus, the ability to integrate/excise the DNA provides flexibility in the design of the constructs.<br>
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<img src="https://static.igem.org/mediawiki/2018/6/61/T--IIT_Delhi--recombinase_action.png" style="border:3px solid #000000" width="48%">
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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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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">1. Conversion between absorbance of cells to the absorbance of a known concentration of beads</b>
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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>
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We at iGEM IIT Delhi made use of serine based recombinases - Bxb1 and TP901 to develop 2  recombinase-based incoherent feed-forward loop constructs. These constructs show a strong capability for regulating and reducing the expression variability of genes being transcribed in addition to faster response time compared to transcriptional networks.
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These constructs can be utilized in the development of complex circuits.
  
<img src="https://static.igem.org/mediawiki/2018/8/8a/T--IIT_Delhi--InterlabParticle2.png" style="border:3px solid #000000" width="48%">
 
<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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">2. Counting colony-forming units (CFUs) from the sample.</b>
 
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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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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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Revision as of 03:18, 18 October 2018

iGEM IIT Delhi



With the advent of synthetic biology, a lot of synthetic circuits are designed every now and then. These circuits contain a lot of components working in harmony to regulate the gene expression. In any system, as the number of components which work together increases, the need for those components to be orthogonal also increases. The components, if interfere with the working of each other, may cause the system as a whole to fail. Such systems succumb to the noise and growth-related factors present in the biological environment. Recombinases in a biological system are adept at this job and hence, are a good basis for the formation of large-scale networks. Utilizing serine recombinases in such biological circuits provides a solution to these problems, as they have non-identical recognition sites and a particular recombinase works orthogonally to other recombinases.

These allow us to build complex circuits with genetic modification in relatively uncomplicated configurations owing to the small size of attB and attP sites(~50bp). The attP and attB sites (and thus the integrase-att complexes) have imperfect two-fold symmetry. Hence they allow recombination in two different ways, with the sites aligned in ‘parallel’ or ‘antiparallel’ configuration. Thus, the ability to integrate/excise the DNA provides flexibility in the design of the constructs.

We at iGEM IIT Delhi made use of serine based recombinases - Bxb1 and TP901 to develop 2 recombinase-based incoherent feed-forward loop constructs. These constructs show a strong capability for regulating and reducing the expression variability of genes being transcribed in addition to faster response time compared to transcriptional networks. These constructs can be utilized in the development of complex circuits.

Contact us

Address

Undergraduate Laboratory
Department of Biotechnology and Biochemical Engineering, IIT Delhi