Part:BBa_I714031

OriT-R (Origin of Transfer for the R751-plasmid nic region)

OriT-R751 (Origin of Transfer for the R751-type conjugative plasmid) is the nic region, where the relaxosome nicks the plasmid and conjugative transfer by R751 plasmid machinery begins via rolling circle replication.

Characterization

//This part was further characterized by repair the point mutation by SUSTech_Shenzhen_2018.

There is a point error(G->A) in No.108bp, which may potentially cause unexpected functional damage. Utilizing artificial mutagenesis by PCR can realize point mutation, so the key point is the primer design. The forward primer is 5’-cgcctcgtctctcatgccggcggtagccggctgcc-3’, and the reverse primer is 5’-gcgaggcagccggctaccgccggcatgagagacgaggcg-3’. The PCR experiment is as follows:

Plasmid_BBa_I714031 2 μL

BM1_mut_F 2 μL

BM1_mut_R 2 μL

2×phanta Max Master Mix 25μL

H2O 19 μL

Total 50 μL

For negative control, the 2×phanta Max Master Mix was replaced by H2O of the same volume.

PCR program was 95°C for 30s, then went to 25 cycles, which included degradation at 95°C for 15s，annealing at 63°C for 15s, extension at 72°C for 2min, then followed by a 5-min 72°C completely extension. Then we run a gel to see whether PCR is successful, which used 15kb marker and 1% gel to run for 30min. Gel image was shown in Figure 1

Figure1. Linearization PCR results of Plasmid_BBa_I714031 First lane: Marker. Second lane: experimental group. Third lane: negative control(NO enzyme)

After that, extraction of DNA sample from gel was done. Then we used DpnI, which cut the methylated GATC sequence, to digest the methylated origin templates and left the newly synthesized DNA. Finally, we directly transformed the digestion product into DH5α, due to the repair system of E.coli, the linearized plasmids was fixed to circular. Then we did miniprep and sent to Sanger sequencing. The sequencing result shown we had successfully generate the point mutation in No.108bp, which means the single error was repaired (Figure 2).

Figure2. Alignment of mutagenesis product and original sequence.

Sequence and Features

Assembly Compatibility:

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

  Illegal NgoMIV site found at 51
  Illegal NgoMIV site found at 104
  Illegal NgoMIV site found at 114
  
• 1000
  COMPATIBLE WITH RFC[1000]