Part:BBa_K5127015:Design

Device for genome integration (pDual-select)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The pDual-select 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. [Image] Figure 1. Plasmid design of pDual-Select. Created by biorender.com.

Results 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). [Image] Figure 2. The Agarose gel electrophoresis result of the PCR products of pReplace construction. A,materials to construct pReplace. B, golden gate assembly result of pReplace construction. The band at 5831bp in (B) indicated the success in plasmid construction.

The plasmid was transformed into E. Coli Trelief 5-alpha for contruction, by plating them on the Kanamycin plates, the construction of this plasmid was being confirmed.

At the same time, the phase I cells were prepared as chemically competnet cells and induced with arabinose for a 3-hour resuscitation. The pDual-Select plasmid was then transformed into these Phase I competent cells, which were subsequently plated on chloramphenicol plates for selection. Only bacteria with successful genome integration of the plasmid will grow on the plate, due to the lost of the second plasmid. (Figure 3.) [Image] Figure 3. The plating result of phase II cells.

Source

E. Coli MG1655