Part:BBa_K118008:Experience

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Characterisation by 2011 iGEM NCTU_FORMOSA

We found a point mutation of Part:BBa_K118008 (iGEM08_Edinburgh) at Spe1 site and we corrected it successfully.(ACCTAGT->ACTAGT) This corrected part is numbered Part:BBa_K539119 The following is the gel electrophoresis result of crtY Part:BBa_K118008. The sample included are uncut plasmid, cut by EcoR1, Xba1, Spe1 and Pst1 respctivly.

We can see that the location of plasmid cut by Spe1 is the same as uncut plasmid. We assumed that Spe1 sit has mutated, so we made the sequence of gene of crtY. Following is the original sequence of crtY Part:BBa_K118008. The SpeI mutate into ACCTAGT.

Therefore, it need to delete the additional amino acid,C. The protocol we used is shown below.
Point Mutation
1.Design primers

GC content: 46.67%                              Location: 168-198
Melting temp: 79.0°C                              Mismatched bases: 1
Length: 30 bp                                      Mutation: Deletion
5' flanking region: 15 bp                           ;Forward primer MW: 9206.10 Da
3' flanking region: 15 bp                           Reverse primer MW: 9206.10 Da

2.Find the best PCR condition by gradient PCR
3.KOD PCR condition

4.Confirm the PCR product with electrophoresis 5.DPN1 37℃ for 3hr~overnight

6.80℃ 20mins to denature the DPN1
7.Confirm with electrophoresis
8.Self ligation room temperature for 2~3 hr

9.Transform DH5alpha with the self-ligation product
Self-ligation product 20 ul
DH5alfa 50 ul
10.Incubate in Ap25 plate then transfer to Ap50 plate.

We correct the Spe1 perfectly. Following is the gel electrophoresis result that show the uncut plasmid and the plasmid cut by Spe1.