We are looking for a person that who can use protein engineering techniques such as rational design; overlap extension metod, whole plasmid method, site directed mutagenesis and non-rational design; error prone PCR, DNA Shuffling, Family DNA Shuffling, StEP, RACHITT, ITCHY, ADO, SCHEMA...
We want to synthesis protein or peptide sequences especially infectious diseases; brucellosis, tuberculosis, Zika Virus and etc..
If someone can provide us new protein or peptide sequences by these methods please contact with me, we will order ..
The basic principles of molecular biology is that you need a gene that codes for a protein. If it is eukaryotic then it is probably glycosylated, you can't do your work in bacteria because you need a Golgi. It is better to not trouble yourself with that so if you are studying bacteria then you should probably use competent Escherichia coli cells.
Determine the gene that codes for your protein. (No introns, please!).
You can scan National Institute for Biotechnology Information for whole genomes: https://www.ncbi.nlm.nih.gov/
You could also reverse translate a protein sequence to the nucleotide sequence. But if the protein has been recombinantly expressed then scientists obviously know the gene.
In the United States, you can order genes, primers, nucleotides and all that stuff from Integrated DNA Technologies. There must be a biology department where you are so they must have some analogous company.
A organism doesn't know that it is supposed to transcribe and translate a gene into a protein if you just throw it into the cell. So you need to put it on a plasmid which has important things like specific antibiotic resistance to like ampicillin, (The common plasmids in molecular biology are the pET vectors.), an inducible polymerase like T7 polymerase which is induced by IPTG or lactose induction. Specifically, you have to insert the gene into your vector in the MULTIPLE CLONING SITE in the pET vectors which the site is flanked by multiple restriction enzyme sites.
This requires cloning:
There are 2 main ways to insert the gene into the multiple cloning site of a plasmid: restriction enzyme ligation and Gibson Assembly.
Restriction enzyme ligation relies on attaching complementary restriction sites to your PCR amplified gene that match the restriction sites on the plasmid. This relies on restriction enzymes that recognize those specific sites and the ligation will be mediated by T4 DNA ligase. So you need to get the PCR right, the DNA extraction from gels right, the restriction enzymes right with right being right according to your system. In order to get the PCR right, you need to use the right primers. In terms of polymerases, Taq makes more mistakes than Phusion because Taq has no proof reading.
Gibson Assembly basically relies on designing primers which overlap with the plasmid site of insertion and also the beginning and end of your gene. Then you apply a DNA polymerase to insert your gene into the multiple cloning site of your plasmid.
Technically, you could digest your plasmid with restriction enzymes to open up the multiple cloning site and then do Gibson Assembly to insert your gene of interest into the multiple cloning site of the plasmid. Anyway, if you get the primers wrong then everything will fail absolutely always.
There is some software that you can use to simulate these experiments in silico so that you know what should work in the lab. I recommend Genome Compiler:
http://www.genomecompiler.com/
Assuming your cloning is done, the next thing that you have to do is transform your plasmid that has your gene in it into a cell. The easiest method is heat shock.
Depending upon what type of cell you just transformed your plasmid into you can produce and extract more of your glorious plasmid (TOP10) or induce protein expression in BL21+ cells with IPTG or lactose induction. Then if your transformation of your plasmid into TOP10 cells worked, you should purify the plasmid from the TOP10 cells and submit the plasmid for sequencing to verify if your gene is in the plasmid and that the plasmid did not self-ligate without the insert.
Now you have conquered the gene and you can work on purifying and characterizing the protein. (If you find that you are unable to fish your protein out of the cell, it may be because you forgot to add an affinity tag such as a polyhistidine tag during the cloning procedure. Don't forget that. )
All this is a couple weeks of hard work.
Anyway, the two best studied proteins are lysozyme and the green fluorescent protein. At my institution these proteins are used to teach undergraduate students how to do these kind of principles of molecular biology. These proteins don't misbehave in the hands of inexperienced. I suggest you look into them, and maybe sign up for a graduate lab class concerning techniques in molecular biology.
I am going to stop talking now ...
That is an interesting research area.
You may would like to check this recent paper:
https://f1000research.com/articles/7-1624
This is entirely bioinformatic work, that is based on analysis of entire E sequences of Zika (particularly, at antigenic sites).
For experimental synthesis, you can design a DNA sequence with desired mutations in silico (e.g.: to increase immunogenicity ..etc), optimize it, and then synthesize it.
Once synthesized, it can be inserted into plasmid (e.g: expression plasmid Pvax1), clone, and purify plasmid DNA.
Then your DNA will be suitable for further studies (In-vitro expression, invite studies, ..etc).
Best,
Iman
Are you also interested in the rational design of new small organic compounds?
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