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

 
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<h1>Assembly Plasmids</h1>
 
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<h1>Assembly Plasmids </h1>
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   <button id="openNav" class="w3-button w3-black w3-xlarge" onclick="w3_open()">&#9776;<p><b>Navigate Results</b></p></button>
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<img src= "https://static.igem.org/mediawiki/2018/9/9f/T--Austin_UTexas--KeyandPlasmid.jpeg" style="width:500px";>
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<figcaption><b>Figure 1.</b> The nine parts that make up every Golden Gate assembly plasmid. Figure taken from "A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly" 2015 Lee, Et. al.</figcaption>
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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> Parts of an Assembly Plasmid </h3>
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<p><b>Assembly plasmids contain an antibiotic resistance gene, origin of replication, origin of transfer, barcode sequence, and a 1-5 bridge part, which contains a colored reporter protein coding sequence.</b> The plasmids are made from 5 different part plasmids: a 1-5 bridge connector and part plasmids 6-8b, using a single BsaI golden gate reaction. The same promoters, terminators, and origins of transfer are used within all assemblies. Each 1-5 bridge connector has a different reporter protein coding sequence making it easy to determine which plasmid each colony contains. Each plasmid also contains a unique Barcode, that is paired to a specific origin of replication. 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. </p>
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<figcaption><b>Table 1.</b> The completed assembly plasmids along with their antibiotic resistance gene, origin of replication, reporter, and barcode.</figcaption>
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<p>We currently have 8 completed assembly plasmids and continue to make more. Table 1 shows the completed assemblies along with which reporter gene, antibiotic resistance gene, origin of replication, and barcode is on the plasmid. All assemblies have the same origin of transfer, assembly connectors, promoter/RBS, and terminator.</p>
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<h2>Shuttle Vector Assemblies</h2>
 
 
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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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Fig 1. BHR 925 Golden Gate Assembly
 
 
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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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<h3> p15A Assembly Plasmid </h3>
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<p> <b>Our lab has created a total of eight assembly plasmids and are constantly working to add more to the kit.</b> Eventually each 1-5 bridge connector, with an assigned chromoprotein reporter and barcode, will be assigned to a specific origin of replication. We currently have created three 1-5 bridge connectors with the GFP, RCP, and E2C reporters. The plate shown below demonstrates in <i> E. coli</i> cells what a successful assembly plasmid transformation looks like with a GFP reporter. While a successfully created part-plasmid does not exhibit any chromoprotein, assembly plasmids do exhibit the chromoprotein from the 1-5 bridge connector part.</p>
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<figcaption><b>Figure 2. Assembly plasmid BHR904</b> This plasmid contains the following parts: 901.1 Bridge connector (GFP), CAM 8b antibiotic resistance, p15A origin of replication, barcode 3, and the origin of transfer.
 
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<p> In figure 2, assembly BHR904 has the 1-5 bridge connector with GFP (901.1) as you can see the single green fluorescent colony on a plate of other non-fluorescent colonies.These non-fluorescent colonies did not pick up the BHR904 plasmid but still have CAM resistance possibly from picking up other undigested part plasmids (all part plasmids have CAM resistance). It is normal to only see a few colony that are transformed with the assembly plasmid and express the chromoprotein</p>
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<h3> Shuttle Vector Assembly</h3>
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<figcaption><b>Figure 3.</b> BHR 925 Golden Gate Assembly</b>
 
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BHR 925 assembly on a LB + CRB plate, viewed under blue light. Red fluorescent colonies (circled) were barely visible after a day of growth but more pronounced after two days.
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<figcaption> Fig 2. 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> 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
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<p>Minpreps of the BHR 922 assemby, which produced colonies but was shown to be only the E2-Crimson  
 
<p>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</p>
 
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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<figcaption>Fig 3. Shuttle Vector Assembly Minipreps Transformed into Top 10 (Left)<br>
 
  
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Left: BHR 925 assembly on a LB + CRB plate<br>
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Top: E2-Crimson 1-5 Bridge plated on a LB + CAM plate<br>
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Right: BHR 922 assembly plated on a LB + KAN plate
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<figcaption><b>Figure 4.</b> 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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<figcaption>Fig 4. BHR 925 Assembly (Left)<br>
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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.
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Latest revision as of 03:45, 18 October 2018


Assembly Plasmids


Figure 1. The nine parts that make up every Golden Gate assembly plasmid. Figure taken from "A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly" 2015 Lee, Et. al.


Parts of an Assembly Plasmid

Assembly plasmids contain an antibiotic resistance gene, origin of replication, origin of transfer, barcode sequence, and a 1-5 bridge part, which contains a colored reporter protein coding sequence. The plasmids are made from 5 different part plasmids: a 1-5 bridge connector and part plasmids 6-8b, using a single BsaI golden gate reaction. The same promoters, terminators, and origins of transfer are used within all assemblies. Each 1-5 bridge connector has a different reporter protein coding sequence making it easy to determine which plasmid each colony contains. Each plasmid also contains a unique Barcode, that is paired to a specific origin of replication. 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.

Table 1. The completed assembly plasmids along with their antibiotic resistance gene, origin of replication, reporter, and barcode.


We currently have 8 completed assembly plasmids and continue to make more. Table 1 shows the completed assemblies along with which reporter gene, antibiotic resistance gene, origin of replication, and barcode is on the plasmid. All assemblies have the same origin of transfer, assembly connectors, promoter/RBS, and terminator.










p15A Assembly Plasmid



Our lab has created a total of eight assembly plasmids and are constantly working to add more to the kit. Eventually each 1-5 bridge connector, with an assigned chromoprotein reporter and barcode, will be assigned to a specific origin of replication. We currently have created three 1-5 bridge connectors with the GFP, RCP, and E2C reporters. The plate shown below demonstrates in E. coli cells what a successful assembly plasmid transformation looks like with a GFP reporter. While a successfully created part-plasmid does not exhibit any chromoprotein, assembly plasmids do exhibit the chromoprotein from the 1-5 bridge connector part.

Figure 2. Assembly plasmid BHR904 This plasmid contains the following parts: 901.1 Bridge connector (GFP), CAM 8b antibiotic resistance, p15A origin of replication, barcode 3, and the origin of transfer.

In figure 2, assembly BHR904 has the 1-5 bridge connector with GFP (901.1) as you can see the single green fluorescent colony on a plate of other non-fluorescent colonies.These non-fluorescent colonies did not pick up the BHR904 plasmid but still have CAM resistance possibly from picking up other undigested part plasmids (all part plasmids have CAM resistance). It is normal to only see a few colony that are transformed with the assembly plasmid and express the chromoprotein

Shuttle Vector Assembly

Figure 3. BHR 925 Golden Gate Assembly
BHR 925 assembly on a LB + CRB plate, viewed under blue light. Red fluorescent colonies (circled) were barely visible after a day of growth but more pronounced after two days.

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


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


Figure 4. 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).
Plate 1: E2-Crimson 1-5 Bridge plated on a LB + CAM plate
Plate 2: BHR 925 assembly on a LB + CRB plate
Plate 3: BHR 922 assembly plated on a LB + KAN plate