Team:Austin UTexas/Results/Assemblies

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Assembly Plasmids

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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

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