Molecule
Golden gate Protocol:
After purification of the PCR product, you can digest your part with EcoRI and PstI using the following Protocol:
Component | Amount (μl) |
---|---|
Purified PCR product | 30 |
EcoRI | 1 |
PstI | 1 |
BsaI | 0.5 |
NEB buffer 4 (10x) | 4 |
ddH2O | 3.5 |
Total Volume | 40 |
Thermocycler programm:
1. 37°C, 12 hours
2. 80°C, 20 minutes
Important note: We very much advise you to digest the vector for 12 hours and purify the product on a gel. This significantly reduces the risk of religation of you vector. We usually had no colonies on our negative control plate after ligation with T4 ligase and transformation into DH10B cells. Since most standard iGEM plasmids contain binding sites of the most common two type IIs restriction enzymes (namely BsaI/Eco31I and BsmBI/Esp3I), we propose using BbsI/BpiI. We have tested this enzyme in various reaction conditions with many different reaction additives (such as ATP or DTT). Although ligase buffer worked best with other type IIs restriction enzymes (in those cases, ligase activity probably was the bottleneck), we had best results with G Buffer (Fermantas) plus several additives using BbsI.
Assembling:
Ligase Reaction:
Plasmid Amplification for further construction;
chose pRSFDuet-1, pACYCDuet-1 and pCDFDuet-1 for expression vector and pBluescript II KS(+) for connector.
Cell
Construct the gene of our fusion protein: ssDsbA-FP-HL-Lgt-FL-TAL using splicing by overlap extension. We connected ssDsbA-FP-HL-lgt for one part of this fusion protein and FL-TAL for another. Then connect them together.
Protein
Connected ssDsbA-FP-HL-lgt by splicing by overlap extension. Transform, colony picking plasmid extraction and digestion identification; Sequencing results showed accurate construction.