Difference between revisions of "Team:Alverno CA/InterLab"

 
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<h1><center>Alverno iGEM Interlab Study</center></h1>
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<center><img src="https://s-media-cache-ak0.pinimg.com/originals/86/08/18/860818cfc65e5ff04725bb0f0c05a8af.png" alt="Alverno iGEM Logo" style="width:300px;"></center>
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    <br>
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        <h2>Interlab Study</h2>
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    </center>
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 +
 
 +
    <center>
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        <h3>Quantifying different sources of variation in gene expression<br> using
 +
        fluorescent transformed E. coli bacteria</h3>
 +
    </center>
 +
 
 +
 
 +
    <div class="demo" id="container">
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        <center>
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            <ul>
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                <li style="list-style: none"><br>
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                <br>
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                <li><img src=
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                "https://static.igem.org/mediawiki/2016/6/6c/T--Alverno_CA--Interlab1.jpg"
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                style="height:215px;width:215px;" title=
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                "Growing bacteria in LB broth">
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                </li>
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                <li><img src=
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                style="height:215px;width:215px;" title=
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                "Looking at plates">
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                <li><img src=
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                style="height:215px;width:215px;" title=
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                "Analyzing data">
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                <li><img src=
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                "Pipetting bacteria to be grown">
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                style="height:215px;width:215px;" title=
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                "Analyzing plate reader data">
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                <li><img src=
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                style="height:215px;width:215px;" title=
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                "Our fluorescent bacteria <br>grown in LB broth">
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                </li>
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                <li><img src=
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                style="height:215px;width:215px;" title=
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                "Diluting cultures">
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                <li><img src=
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                style="height:215px;width:215px;" title=
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                "Pipetting FITC standard curve">
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                <li><img src=
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                style="height:215px;width:215px;" title=
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                "Plate reader scan for ODs">
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 +
 
 +
    <h5>
 +
    </h5>
 +
 
 +
 
 +
    <h4>Summary:</h4>
 +
    <h5></h5>
 +
 
 +
    <h5>As part of the 2016 iGEM Interlab Study, we tested 3 different
 +
    fluorescent protein expression plasmids, J23101, J23106, and J23117 with
 +
    positive and negative controls. We transformed the parts with iGEM's
 +
    transformation protocol. Our bacteria were cultured on a LB agar plate and
 +
    cultured in LB broth. To measure the differences in fluorescence expressed
 +
    by different strength plasmids, we used a plate reader with an OD600
 +
    reference point and FITC standard curve.</h5>
 +
 
 +
 
 +
    <h5>
 +
    </h5>
 +
<br><br>
 +
 
 +
    <h4>Project Description:</h4>
 +
    <h5></h5>
 +
 
 +
    <h5>The 2016 iGEM Interlab Study aims to compare results obtained by various
 +
    teams in order to quantify the expression of 5 different constructs using
 +
    fluorescence, which provides a useful insight into expression levels that
 +
    can be monitored without disrupting cells. Each device used in the study is
 +
    in the pSB1C3 plasmid backbone and are fluorescent protein expression
 +
    plasmids of different strength promoters. The devices we tested were
 +
    J23101, J23106, J23117 (Test Devices 1, 2, and 3). We used positive control
 +
    I20270 and negative control R0040. J23101, with a strong promoter, showed
 +
    the highest amount of fluorescence. J23117, with a weak promoter, showed
 +
    the lowest amount of fluorescence, and J23106, with the medium strength
 +
    promoter, showed fluorescence amounts between the other two. Our test
 +
    devices were inserted into DH5alpha E. coli, which were transformed
 +
    according to the iGEM transformation protocol. Our transformed cells were
 +
    plated on LB plates with chloramphenicol and incubated overnight at 37° C.
 +
    Two colonies were picked from each plate (with the exception of Test Device
 +
    3, with which there was only one) and were inoculated in 15mL test tubes.
 +
    These devices were diluted, and then measured in a plate reader with an
 +
    excitation wavelength of 35nm, according to iGEM’s measurement
 +
    protocol.</h5>
 +
 
 +
 
 +
    <h5>
 +
    </h5>
 +
 
 +
 
 +
    <h5>
 +
    </h5>
 +
<br><br>
 +
 
 +
    <h4>Protocol:</h4>
 +
    <h5></h5>
 +
 
 +
    <h5><a href=
 +
    "https://static.igem.org/mediawiki/2016/c/c5/InterLab_iGEM2016_Plate_Reader_Protocol_Updated_July.pdf">
 +
    Plate Reader Protocol</a>
 +
    </h5>
 +
 
 +
 
 +
    <h5><a href=
 +
    "https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf">
 +
    Transformation Protocol</a>
 +
    </h5>
 +
 
 +
 
 +
    <h5>
 +
    </h5>
  
−
<h1><center>Our Project</center></h1>
+
<br><br>
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<h2><center>CRISPR/Cas9 Clamp to Block propagation of Supercoiling</center></h2>
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    <h4>Results:</h4>
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<h2><center>Generated during Transcription</h2></center>
+
<h5>You can download our data sheet <a href="https://goo.gl/uofZIO">here.</a></h5>
−
<h3>Executive Summary:</h3>
+
−
<p>    We propose using deactivated Cas9 (dCas9) as a DNA clamp to block propagation of supercoiling generated during transcription, improving the modularity, predictability, and scalability of single-vector, multi-gene synthetic systems.</p>  
+
  
−
<p><h3>Project Description:</h3></p>
+
<img class:icons src="https://static.igem.org/mediawiki/2016/0/00/T--Alverno_CA--interabs.png" alt="Abs600" style="width:800px;">
−
<p>   Synthetic biology is best pursued with orthogonal, composable parts. For example, when a promoter is characterized, it should perform predictably no matter what gene or UTR it is composed with (at least under the same environmental conditions). This form of orthogonality is generally assumed to hold, and is the basis for most models of part characterization and composition.</p>
+
<h5>Graph of Absorbance (600) over a 6 hour time period.
−
<p>   One context in which orthogonality is demonstrably broken is composition of multiple independent gene expression unit on the same piece of linear, genomic, or plasmid DNA. When two fully-characterized expression units (say, for example, a GFP coding sequence with promoter, RBS, and terminator, and an RFP coding sequence with promoter, RBS, and terminator) are placed next to each other on the same plasmid, their expression levels will be different than if they had been placed on different plasmids[1]. Worse, the relative orientation of the two genes dramatically affects expression. These effects are difficult to predict, so multi-gene systems typically must be optimized using time-consuming screens to achieve desired expression levels (or are not optimized at all).</p>  
+
</h5>
−
<p>   It is not known for certain why compositional context affects gene expression, but one promising hypothesis involves DNA supercoiling. The process of transcription torques DNA near the transcription site, introducing supercoils that can propagate down the DNA[2]. Supercoiling state, in turn, is known to affect transcription, with negative and positive supercoils making transcription more and less efficient, respectively[3]. Taken together, these effects suggest a mechanism by which genes on the same plasmid can affect each other’s expression levels[1].</p>
+
<br><br>
−
<p>   In our proposed project, we will attempt to isolate the expression of genes on the same plasmid (or other piece of DNA) by using DNA binding proteins as “clamps” to block propagation of supercoiling. By placing a recognition sequence for one of several DNA binding proteins (i.e., tetR, cI, etc.) between two expressing genes (and possibly another on the opposite side of the plasmid), we hope to block the propagation of supercoils between those genes, thereby isolating their expression.</p>
+
<img class:icons src="https://static.igem.org/mediawiki/2016/c/ca/T--Alverno_CA--interfl.png" alt="Fl" style="width:800px;">
−
<p>    As one of the stronger known DNA binding proteins, Cas9 is an ideal candidate for a DNA-binding clamp. Specifically, we will use dCas9, a version of Cas9 engineered to bind, but not cut, DNA. In addition to being a strongly-binding clamp, dCas9 has the advantage of not being used naturally as a transcription factor; unlike a naturally-occurring repressor such as tetR or lacI, dCas9 can be used to isolate contiguous gene constructs without otherwise affecting host gene expression.</p>
+
<h5>Graph of Fluorescence in AU over a 6 hour time period.
−
<p>   To test dCas9 as a clamp, we will clone several plasmids each containing two different fluorescent reporters, in different relative orientations, with several dCas9 binding sites between them. We will express these plasmids both in vivo and in an in vitro gene circuit prototyping system alongside a dCas expression plasmid and gRNA expression plasmids targeting zero or more clamp sites. We expect expression levels of the reporter proteins to vary by orientation when no gRNA is expressed, but for those differences to be at least partially diminished when one or more targeting gRNAs are expressed.</p>  
+
</h5>
−
​
+
<br><br>
−
<h3>Potential Applications and Implications:</h3>  
+
<img class:icons src="https://static.igem.org/mediawiki/2016/8/82/T--Alverno_CA--interflabs2.png" alt="Fl/Abs" style="width:800px;">
−
<p>    If successful, a dCas9-based isolating clamp could be used in any multi-gene circuit assembly, making multi-gene assemblies more predictable and their assembly much more efficient.</p>  
+
<h5>Graph of Fluorescence/Absorbance in AU (log scale) over a 6 hour time period. Amounts of Fl/Abs600 are consistent with the strengths of the promoters.
−
<p>   This is particularly relevant in metabolic engineering, where circuits of many genes are routinely constructed. The physical layout of these circuits can unpredictably affect production of output by several orders of magnitude[4], so large engineered metabolic pathways must typically be hand-tuned or have many configurations screened for activity. By making gene expression more predictable, our results could greatly improve the predictability (and, therefore, designability) of large gene circuits for metabolic engineering.</p>
+
</h5>
 +
<br><br>
 +
<img class:icons src="https://static.igem.org/mediawiki/2016/c/c2/T--Alverno_CA--interfitc.png" alt="FITC" style="width:800px;">
 +
<h5>FITC Fluorescence standard curve. Row A had pipetting errors and so is different from the other curves.
 +
</h5>
 +
<br>
 +
 
  
−
<h3>References:</h3>
 
−
<p> 1. Yeung, E. 2016. Reverse Engineering and Quantifying Context Effects in Synthetic Gene Networks [dissertation]. [Caltech (CA)]: Caltech.</p>
 
−
<p> 2. Tsao, Y., Wu, H., Liu, L. F. 1989. Transcription-Driven Supercoiling of DNA: Direct Biochemical Evidence from In Vitro Studies. Cell. 56:111-118.</p>
 
−
<p> 3. Hanafi, E.D., Bossi, L. 2000. Activation and silencing of leu-500 promoter by transcription-induced DNA supercoiling in the Salmonella chromosome. Mol Microbiol. 37(3):583-94.</p>
 
−
<p> 4. Smanski, M. J., Bhatia, S., Zhao, D., Park, Y., Woodruff, L. B. A., et al. 2014. Functional optimization of gene clusters by combinatorial design and assembly. Nature Biotechnology. 32:1241-1249.</p>
 
  
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Latest revision as of 03:32, 20 October 2016

Alverno iGEM 2016


Interlab Study

Quantifying different sources of variation in gene expression
using fluorescent transformed E. coli bacteria































Summary:

As part of the 2016 iGEM Interlab Study, we tested 3 different fluorescent protein expression plasmids, J23101, J23106, and J23117 with positive and negative controls. We transformed the parts with iGEM's transformation protocol. Our bacteria were cultured on a LB agar plate and cultured in LB broth. To measure the differences in fluorescence expressed by different strength plasmids, we used a plate reader with an OD600 reference point and FITC standard curve.


Project Description:

The 2016 iGEM Interlab Study aims to compare results obtained by various teams in order to quantify the expression of 5 different constructs using fluorescence, which provides a useful insight into expression levels that can be monitored without disrupting cells. Each device used in the study is in the pSB1C3 plasmid backbone and are fluorescent protein expression plasmids of different strength promoters. The devices we tested were J23101, J23106, J23117 (Test Devices 1, 2, and 3). We used positive control I20270 and negative control R0040. J23101, with a strong promoter, showed the highest amount of fluorescence. J23117, with a weak promoter, showed the lowest amount of fluorescence, and J23106, with the medium strength promoter, showed fluorescence amounts between the other two. Our test devices were inserted into DH5alpha E. coli, which were transformed according to the iGEM transformation protocol. Our transformed cells were plated on LB plates with chloramphenicol and incubated overnight at 37° C. Two colonies were picked from each plate (with the exception of Test Device 3, with which there was only one) and were inoculated in 15mL test tubes. These devices were diluted, and then measured in a plate reader with an excitation wavelength of 35nm, according to iGEM’s measurement protocol.


Protocol:

Plate Reader Protocol
Transformation Protocol


Results:

You can download our data sheet here.
Abs600
Graph of Absorbance (600) over a 6 hour time period.


Fl
Graph of Fluorescence in AU over a 6 hour time period.


Fl/Abs
Graph of Fluorescence/Absorbance in AU (log scale) over a 6 hour time period. Amounts of Fl/Abs600 are consistent with the strengths of the promoters.


FITC
FITC Fluorescence standard curve. Row A had pipetting errors and so is different from the other curves.