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

 
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                 "Liquid cultures of RFP/GFP constructs; transformed <i>E. coli</i>">
 
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<h3>Introduction </h3>
 
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<h5> We believed that supercoiling has been a problem for synthetic biologists trying to construct plasmids expressing multiple genes. We created plasmids to demonstrate supercoiling and the interference it creates between two genes. Our plasmids contain one GFP coding device and one RFP coding device, in varying orientations, with either a 500bp spacer, 1000bp spacer, or dCas9 clamp binding sites between them. The plasmids use the backbone pSB1C3 or <a href="https://www.addgene.org/66067/"> DVK_AE</a> (derived from pSB1K3). Because the GFP and RFP devices are placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (For more details, see <a href="https://2016.igem.org/Team:Alverno_CA/Design">Design</a>) After creating successful plasmids, we transformed them in E. coli. The colonies were then inoculated and grown as overnight liquid cultures. </p>
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<h5> It has been reported in the synthetic biology literature that the relative orientations and positions of genes physically placed near each other in multigene circuits may affect their expression. Specifically, it has been postulated that the issue of supercoiling may be responsible for this interference. Considering this, our intent was to demonstrate these effects on a series of plasmids containing both a RFP coding device, and a GFP coding device on a pSB1C3 pSB1C3 or <a href="https://www.addgene.org/66067/">DVK_AE</a> (derived from pSB1K3). In between the two genes on each plasmid, we placed one of several insulators to separate the genes. These included a 500bp spacer, a 1000 bp spacer, and a dCas9 clamp; each was designed to isolate and eliminate interference. Constructed plasmids were inserted into E. coli (DH5α). Ultimately, the goal of the experiment was to accurately demonstrate interference between two genes (RFP/GFP) on the same plasmid and, in addition, find an insulating mechanism to restrain the effects of possible supercoiling. (For more details, see <a href="https://2016.igem.org/Team:Alverno_CA/Design">Design</a>)  
  
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<br>After constructing these plasmids, we grew transformed <i>E. coli</i> as overnight liquid cultures. </h5>
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<h4>Methods </h4>
 
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<h4> For in vivo using liquid cultures</h4>
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<h3>Testing spacers <i>in vivo</i></h3>
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<h5> For cultures that containing strains with plasmids that had a 500bp spacer or 1000bp spacer, we diluted these cultures to normalize them to the same OD absorbance. Then, we measured RFP expression (in AFU), GFP expression(in AFU), and OD (absorbance) in a plate reader (<a href="http://www.perkinelmer.com/product/victor-x5-for-fl-lum-uv-trf-fp-2030-0050">VICTOR-X3</a>). For strains using a 1000bp spacer we tried testing the effects of using inducer (ATc and IPTG) as well. Using these results we were able to analyze the effects of supercoiling and the effects of using a base-pair spacer between them in order to reduce supercoiling. (See our <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">Plate Reading (for Fluorescence, Absorbance, Induction, etc.) Protocol</a> for more information) </h5>
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<h5>Cultures of strains containing plasmids with a 500bp spacer or 1000bp spacer were diluted to the same OD. RFP expression (in AFU), GFP expression (in AFU), and OD were measured using a <a href="http://www.perkinelmer.com/product/victor-x5-for-fl-lum-uv-trf-fp-2030-0050">VICTOR-X3</a> plate reader. Inducers - specifically ATc and IPTG - were added to the strains containing plasmids with the 1000bp spacer. (See <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">"Plate Reading"</a> for more information) </h5>
  
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<h3> Testing dCas9 clamps <i>in vitro</i> </h3>
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<h5> Cultures of strains containing plasmids with a dCas9 clamp site spacer were mini-prepped to extract plasmids. We designed and constructed gRNA plasmids and obtained dCas9 expression plasmids (<a href="https://www.addgene.org/48657/">DS-SPCasN</a>). All plasmids were mini-prepped and expressed in TX-TL <a href="http://www.openwetware.org/wiki/Biomolecular_Breadboards">TX-TL</a>, an <i>in vitro</i> prototyping technique which mimics cell environments for transcription and translation. The plasmids were tested with and without inducers (IPTG for RFP, and ATc for GFP). GFP and RFP were measured with the plate reader. (See <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">"Plate Reading"</a> for more information)</h5>
  
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<h4> For in vitro using liquid plasmid DNA by TX-TL </h4>
 
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<h5> For cultures containing strains with plasmids that had a dCas9 clamp site spacer, cultures were mini-prepped to extract the plasmid DNA. In order to run our plasmids containing the dCas9 clamp site, we also had to design gRNA plasmids and obtain dCas9 expression plasmids. (<a href="https://www.addgene.org/48657/">DS-SPCasN-</a>)). All three of these plasmids were miniprepped and then expressed using TX-TL, an in vitro, prototyping technique which mimics cell environments for transcription and translation. For our first run we did not use inducer, but for our second run we used ATc and IPTG to induce the TetR and LacI promoters on the GFP and RFP devices, respectively. In the plate reader we obtained results that measured GFP and RFP expression. (see <a href="https://2016.igem.org/Team:Alverno_CA/Results">Results</a>) Once again, because the GFP and RFP devices were placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (See our <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">Plate Reading (for Fluorescence using TX-TL for GG105-108 w/ dcas9 expression plasmids) Protocol</a> for more information)</h5>
 
  
 
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Latest revision as of 20:02, 2 November 2016


Experiment













Introduction

It has been reported in the synthetic biology literature that the relative orientations and positions of genes physically placed near each other in multigene circuits may affect their expression. Specifically, it has been postulated that the issue of supercoiling may be responsible for this interference. Considering this, our intent was to demonstrate these effects on a series of plasmids containing both a RFP coding device, and a GFP coding device on a pSB1C3 pSB1C3 or DVK_AE (derived from pSB1K3). In between the two genes on each plasmid, we placed one of several insulators to separate the genes. These included a 500bp spacer, a 1000 bp spacer, and a dCas9 clamp; each was designed to isolate and eliminate interference. Constructed plasmids were inserted into E. coli (DH5α). Ultimately, the goal of the experiment was to accurately demonstrate interference between two genes (RFP/GFP) on the same plasmid and, in addition, find an insulating mechanism to restrain the effects of possible supercoiling. (For more details, see Design)
After constructing these plasmids, we grew transformed E. coli as overnight liquid cultures.


Methods


Testing spacers in vivo

Cultures of strains containing plasmids with a 500bp spacer or 1000bp spacer were diluted to the same OD. RFP expression (in AFU), GFP expression (in AFU), and OD were measured using a VICTOR-X3 plate reader. Inducers - specifically ATc and IPTG - were added to the strains containing plasmids with the 1000bp spacer. (See "Plate Reading" for more information)

Testing dCas9 clamps in vitro

Cultures of strains containing plasmids with a dCas9 clamp site spacer were mini-prepped to extract plasmids. We designed and constructed gRNA plasmids and obtained dCas9 expression plasmids (DS-SPCasN). All plasmids were mini-prepped and expressed in TX-TL TX-TL, an in vitro prototyping technique which mimics cell environments for transcription and translation. The plasmids were tested with and without inducers (IPTG for RFP, and ATc for GFP). GFP and RFP were measured with the plate reader. (See "Plate Reading" for more information)

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