overexpression xylanase gene

[nekonara]

i'm currently in practical training. my supervisor given me a project to conduct. can u help me understanding this project?

overexpression of xylanase gene of bacillus (mutant and wild type) into e coli

1. how can i perform this experiment? step by step if you may

2. how many type of vectors i have to use for this experiment?

3. how to check the activity of expressed gene?

4. what is mean by gene expression. what i really need to do by overexpression. how to over express the gene.

thank you for spending your time

[JackBean]

1) this can have several guidelines, since bacteria have no introns, you can easily clone the gene directly from genomic DNA. This clone into any vector (either one for keeping the gene or directly into overexpression one). Transform the vector into E. coli and express.
2) from one to several
3) either detect mRNA (is there any protocol for that in bacteria? Probably yes.), detect protein e.g. via Western blot or measure activity if is it possible
4) basically any expression with artificial promotor is overexpression

[nekonara]

thank you mr jack bean.

i'm thinking about using pRSET vector for overexpression. and spread plate of LB + amp to select the transformants. i think all metods step by step i'd get from my research in the net and from other sources.

one thing that give me a headache is how i have to do to check the activity. all i know is i will compare it to non transformed. my supervisor had told me 2 clues (before he went to conference of microbiology - and he is unreachable) that i have to do batch fermentation and *something* PCR. i didnt know either he's talking about real time PCR or reverse transcriptase PCR.

what do you think about the clues? how can i relate it to what i have to do to check the activity of over expressed transformant?

thank you again for helping