Engineering a recombinant plasmid DNA(like GST- preferably use other than GST)to over-express the protein encoded by the MsrB gene from Drosophila, by cloning it into the pGEX vector. the purpose of this assignment is to allow to design our own similar approach for overexpression of a recombinant protein of our own choosing.
1-select one Drosophila gene that you would like to use for this project. FlyBase.org is a database of Drosophila genes and is therefore a good place to start, however we can obtain our gene from another source if we prefer.
2-find the DNA sequence for the open-reading frame (ORF) of our selected gene. this is the portion of the mRNA that codes for the amino acids. It will start with an ATG(AUG) 'start codin, and end with one of the three 'stop' codons (TAG,TAA,TGA), often, the complete gene sequence will be shown with the exon highlighted.
3-design PCR primers between 25-30 nucleotides in length that can be used to amplify the cDNA and allow cloning into the pGEX-4T-2 vector. as shown on the map of the pGEX vector, we can use BamHI,EcoRI,SmaI,SalI,XhoI or NotI for cloning.
4-show the cDNA sequence and corresponding primer sequence for both the upstream and downstream primers. be very clear about any base changes in the primer that are needed to create the restriction site and or maintain the correct reading frame.
5-determine the amino acid sequence that precedes the Met start codon after the fusion protein is cleaved with thrombin.
IMPORTANT POINTS TO KEEP IN MIND:
1-the cDNA must be inserted in the correct orientation. the 5' end must be closest to the thrombin cleavage site.
2-our PCR amplicon cannot contain a recognition site for either of the enzymes that we have chosen for cloning (why not?)
3-we must maintain the proper reading frame. note how bases can be added or delete from the 5' upstream primer as needed.
4-try to clone the cDNA as close to the start codon as possible.
5-we do not have to worry about the reading frame for the downstream primer, but be sure that the stop codon of the cDNA is retained.
1. Gene of interest. I like Wingless (http://flybase.org/reports/FBgn0004009.html).
2. On the flybase page I selected the fasta format of the coding region sequence:
Several paragraphs with web references detailing how to design primers to clone a gene of interest with restriction enzyme cut sites added to the primer. Consideration is given for orientation and frame.