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.

As of now we have completed 9 assembly plasmids and hope to keep adding to the list. Figure 2 to the left shows a table of all the completed assemblies along with which chromoprotein, antibiotic resistance, origin, and barcode is on the 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

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

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. 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