Team:CHINA CD UESTC/Method
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METHOD
We present details on the various methods such as vectors design, domain linker selection and choose of enzyme insertion site that used in the experiment on this page, if you are willing to check or follow our work, you can scan the methods here. Any questions or advice to us are acceptable at any time.
If you want to check or follow our project, you can read the METHOD page to get main information concerning our project. In addition, you will get more details about experiment from our protocols.
Q1: How to get target gene?
Four gene clusters related to magnetosome synthesis(mamAB/mamGFDC/mamYXZ/mms6) and mamW: use the magnetotactic bacteria’s (Magnetospirillum gryphiswaldense MSR -1) genome as template to amplify.
Laccase gene and RFP: use the DNA fragments from 2015 Kit Plate2 provided by iGEM (Name: BBa_K863005, BBa_E1010) and amplify them through common PCR.
Q2: Where are the backbone vectors from?
All backbone vectors are purchased from Biotech Corp. They are pET28a, pCDFDuet-1 and pACYCDuet-1, the first two aim to carry gene clusters that realize magnetosome generating and the last one is for putting together the genes of laccase + mamW + RFP.
Q3: What kinds of linker we chose for linking between our protein domains?
We obtained the linker information through searching in the Registry of Standard Biological Parts, finally we used two kinds of linkers.
•ggtggaggaggctctggtggaggcggtagcggaggcggagggtcg
Same as the (Gly4Ser)3 Flexible Peptide Linker (Name: BBa_K416001): between mamW, RFP and Laccase.
•gcaggtagcggcagcggtagcggtagcggcagcgcg
Refer to 6aa [GS]x linker(Name: BBa_J18921): between mamW and RFP.
Q4: What kinds of enzymes we used when we made target gene insert into vectors?
•pET28a:
For consideration of the gene cluster size (17kb), we divided mamAB operon into three parts, we used (ApaⅠ)(SapⅠ)(ArvⅡ)(NotⅠ) to come true the insertion of mamAB.
•pCDFDuet-1:
The restriction enzyme cut sites flanking mamGFDC+mms6 on either side are (HindⅢ) and (XhoⅠ), flanking mamYXZ on either side are (XbaⅠ)and (PstⅠ)
•pACYCDuet-1:
The restriction enzyme cut sites flanking Laccase + mamW + RFP on either side are (PstⅠ) and (XhoⅠ).