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

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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>
 
<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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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.  
 
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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Revision as of 17:34, 14 October 2018


Assembly Plasmids


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.

Shuttle Vector Assemblies


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

Fig 1. BHR 925 Golden Gate Assembly
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.
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.

Fig 2. Sequence alignment of BHR 925 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.

Minpreps of the BHR 922 assemby, which produced colonies but was shown to be only the E2-Crimson 1 – 5 bridge, was re-transformed into E. coli along with BHR 920 and BHR 925 to better view expression


Fig 3. Shuttle Vector Assembly Minipreps Transformed into Top 10 (Left)
Left: BHR 925 assembly on a LB + CRB plate
Top: E2-Crimson 1-5 Bridge plated on a LB + CAM plate
Right: BHR 922 assembly plated on a LB + KAN plate
Fig 4. BHR 925 Assembly (Left)
BHR 925 assembly miniprep transformed into Top 10 cells and plated on a LB + Agar plate. Viewed under visible light the dark blue colonies are clearly present.