Part:BBa_K5127025
Device for genome integration (pReplace)
This plasmid is the third plasmid we constructed for genome integration. pReplace was designed to facilitate the integration of the Gene of Interest (GOI) into the genome.
Usage and Biology
On either side of the GOI (here we have a barcode instead of GOI) there are two homology arms, HA Rev and HA For, each approximately 500bp long. These homology arms allow for precise recombination into the genome, mediated by integrase and recombinase enzymes. The integration site and the homology arms were derived from Park, Y. et al (2020). After transforming the pReplace plasmid into the Phase II competent cells (BBa_K5127015), the barcode is automatically integrated into the genome. At the same time, the bacteria are plated on 10% sucrose plates, and only those that have successfully replaced the SacB gene with the barcode will survive, as SacB is lethal in the presence of sucrose. The colonies that grow on these plates indicate successful integration. Finally, we will confirm the integration through colony PCR.
Team: BNDS-China 2024
Design
The pReplace plasmid was assembled with an R6K origin of replication (ori), which can only replicate in E. coli DH5-alpha pir+ strain. This design ensures that when the plasmid is transformed into E. coli MG1655, they cannot replicate, preventing the presence of redundant plasmid copies and avoiding false-positive results during integration verification. In addition, the plasmid contains homology arms to guide presice integration of the GOI into the specific, non-essential regions of the E. Coli MG1655's genome. This ensures that the integration occurs at safe sites, avoiding disruption of the organisms' basic functions and maintaining normal cellular processes (Park et al.,2020). Finally, by sequentially transforming the three plasmids, it is possible for us to integrate the GOI into the E. Coli MG1655's genome. (See our protocol for detailed steps).
Figure 1. Plasmid design of pReplace. Created by biorender.com.
Build
The sequence of homology arms and R6K Ori were obtained from BNDS-China's previous plamids. We used Golden Gate Assembly to construct pReplace. PCR and Gel Electrophoresis were performed to verify the success in constructing the fragment and backbone of the full plasmid (Figure 2).
Figure 2. The Agarose gel electrophoresis result of the PCR products of pReplace construction. A, B,materials to construct pReplace. C, golden gate assembly result of pReplace construction. The band at 2932bp in (C) indicated the success in plasmid construction.
Result
After transforming pReplace into Phase II competent cells, we initially observed a bacterial lawn on the plate (Figure 3A). After incubating the plate at 37 degree Celcius for three days, we identified single bacterial colony (Figure 3B). Then, we inoculated colonies and performed colony PCR, which showed the correct length, indicating successful integration (Figure 3C). However, due to limitations in sequencing capabilities in our lab, we were unable to obtain sequencing results for successful PCR products.
Figure 3. Observation and PCR verification of Phase III cells. A, The bacterial lawn of Phase III cells. B, The single colonies occur after being cultured at 37 degree Celsius for several days. C, The AGE result for Phase III colony PCR verification. The band at 1753bp indicates a successful amplification from genome.
Next, we plan to apply this method to integrate Tes4 into the genome of Nissle 1917. For more details, refer to (BBa_K5127015)(BBa_K5127016). This will allow us to extend our platform's functionality and explore Tes4's role in butyrate production and its effects on probiotic growth.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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