Difference between revisions of "Team:Austin UTexas/Results/Assemblies"
Alyssab222 (Talk | contribs) |
Alyssab222 (Talk | contribs) |
||
| Line 161: | Line 161: | ||
<figcaption> <font size="3"><b>Figure 1.</b> Shows the nine parts that make up every assembly plasmid</font> </figcaption> | <figcaption> <font size="3"><b>Figure 1.</b> Shows the nine parts that make up every assembly plasmid</font> </figcaption> | ||
</figure> | </figure> | ||
| − | + | <br> | |
</div> | </div> | ||
<br> | <br> | ||
| − | + | <h4> p15A Assembly Plasmid</h4> | |
| + | <br> | ||
| + | <br> | ||
<div class= "floatright"> | <div class= "floatright"> | ||
| Line 171: | Line 173: | ||
<figure> | <figure> | ||
| − | <img src = "https://static.igem.org/mediawiki/2018/b/b3/T--Austin_UTexas--AnnotatedCAMplate.jpg" style="width: | + | <img src = "https://static.igem.org/mediawiki/2018/b/b3/T--Austin_UTexas--AnnotatedCAMplate.jpg" style="width:300px;> |
<figcaption> | <figcaption> | ||
| Line 179: | Line 181: | ||
</div> | </div> | ||
| + | <h4> Shuttle Vector Assembly</h4> | ||
| + | <br> | ||
| + | <br> | ||
| + | <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> | ||
| + | <figure> <img class="resize" src = "https://static.igem.org/mediawiki/2018/0/0e/T--Austin_UTexas--ShuttleVectorAssembly1.jpeg" > | ||
| + | <caption align="bottom"> | ||
| + | <b>Fig 1. BHR 925 Golden Gate Assembly</b> | ||
| + | <br> | ||
| + | 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. | ||
| + | <br> | ||
| + | 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. | ||
| + | |||
| + | </figcaption> | ||
| + | |||
| + | </figure> | ||
| + | |||
| + | |||
<script> | <script> | ||
Revision as of 07:40, 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.
Figure 1. Shows the nine parts that make up every assembly plasmid
p15A Assembly Plasmid
Our lab has created a total of nine 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. 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.
Figure 2. Assembly plasmid BHR904. 901.1 Bridge connector (GFP), CAM 8b antibiotic resistance, p15A origin of replication, barcode 3, and origin of transfer
Shuttle Vector Assembly
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
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.