Difference between revisions of "Team:Austin UTexas/Results/Assemblies"

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<h3> Assembly Plasmids </h3>
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<p>Using five single parts and one bridge-type part, full plasmids are assembled in a single BsaI reaction. An assembled plasmid then contains an antibiotic resistance gene, origin of replication, origin of transfer, barcode sequence, and a part 1-5 bridge, which contains a colored reporter protein coding sequence. The same promoters, terminators, and origins of transfer are used within all assemblies. This is not true for the barcode, and reporter protein sequences, as they indicated which origin of replication the plasmid contains. This design eliminates as many variables as possible, allowing the origin of replication to be the single largest factor determining whether or not the bacteria sustain and express the plasmid. </p>
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<h3>Shuttle Vector Assemblies</h3>
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<p>Broad Host Range assemblies encoding a shuttle vector, pAMBI, were assembled using standard Golden Gate assembly protocols. BHR 925 (E2-Crimson + pAMBI Origin + CRB resistance), 920 (E2-Crimson + pAMBI Origin + KAN resistance), and 922 produced darker, bluish colonies under visible light. When viewed under a UV light, the colonies were red, indicating the successful expression of E2-Crimson</p>
 
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<figure> <img class="resize" src = "https://static.igem.org/mediawiki/2018/0/0e/T--Austin_UTexas--ShuttleVectorAssembly1.jpeg">
 
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<figcaption>
 
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<b>Fig 1. BHR 925 Golden Gate Assembly</b>
 
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Left: BHR 925 assembly on a LB + CRB plate, viewed under blue light. Red fluorescent colonies were barely visible after a day of growth but more pronounced after two days.
 
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Right: BHR 925 assembly viewed under visible light. Dark grey, blue colonies were barely visible after a day of growth but color expressed more clearly after a few days. Colonies were picked into liquid culture. Cultures which became blue were miniprepped and sequenced.
 
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<figure><img class="resize" src = "https://static.igem.org/mediawiki/2018/c/c6/T--Austin_UTexas--ShuttleVectorAssembly2.png">
 
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<figcaption> <b>Fig 2.</b> Sequence alignment of BHR 925
 
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Sequencing confirmed the BHR 4 barcode, BHR 701 (origin of transfer), and forward portion of the pAMBI origin were present in the BHR 925 assembly. Sequencing was inconclusive for BHR 922 but colonies grew on KAN plate and expressed E2- Crimson.
 
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<p>Minpreps of the BHR 922 assemby, which produced colonies but was shown to be only the E2-Crimson
 
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1 – 5 bridge, was re-transformed into E. coli along with BHR 920 and BHR 925 to better view expression</p>
 
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<figure>
 
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    <img class="resize" src="https://static.igem.org/mediawiki/2018/4/43/T--Austin_UTexas--shuttlevectorfigure.png">
 
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<figcaption>Fig 3. BHR 925 Shuttle Vector Assembly Transformed into Top 10. Viewed under visible light the dark blue colonies are clearly present(Left) Shuttle Vector Assembly Minipreps Transformed into Top 10 (Right)
 
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Plate 1: E2-Crimson 1-5 Bridge plated on a LB + CAM plate<br>
 
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Plate 2: BHR 925 assembly on a LB + CRB plate<br>
 
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Plate 3: BHR 922 assembly plated on a LB + KAN plate
 
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Assembly plasmids are assembled using a 1-5 bridge connector and part plasmids 6-8b in a single BsaI golden gate reaction. An assembled plasmid then contains an antibiotic resistance gene, origin of replication, origin of transfer, barcode sequence, and a part 1-5 bridge, which contains a colored reporter protein coding sequence. The same promoters, terminators, and origins of transfer are used within all assemblies. Each 1-5 bridge connector has a different barcode and reporter protein coding sequence making it easy to determine which plasmid each colony contains. This design eliminates as many variables as possible, allowing the origin of replication to be the single largest factor determining whether the bacteria sustain and express the plasmid. Using golden gate assembly, we were able to assemble these plasmids in just one reaction with the help of overhangs common to each part type.
  
 
<script>
 
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Revision as of 05:37, 16 October 2018


Assembly Plasmids


Assembly Plasmids


Assembly plasmids are assembled using a 1-5 bridge connector and part plasmids 6-8b in a single BsaI golden gate reaction. An assembled plasmid then contains an antibiotic resistance gene, origin of replication, origin of transfer, barcode sequence, and a part 1-5 bridge, which contains a colored reporter protein coding sequence. The same promoters, terminators, and origins of transfer are used within all assemblies. Each 1-5 bridge connector has a different barcode and reporter protein coding sequence making it easy to determine which plasmid each colony contains. This design eliminates as many variables as possible, allowing the origin of replication to be the single largest factor determining whether the bacteria sustain and express the plasmid. Using golden gate assembly, we were able to assemble these plasmids in just one reaction with the help of overhangs common to each part type.