I am trying to knockout a bacterial gene by homologous recombination. I have designed a knockout construct having 500 upstream, 500 downstream, 100 bp of each 5` and 3` end of the gene with a restriction site in between. After homologous recombination the knockout gene will contain 208 bp from ATG to TAA which is not in triplet frame. Will this affect transcription of adjacent gene.
Yes, it can affect the transcription of downstream genes if the open reading frame you are deleting is co-transcribed with others. However, it depends on how you knock-out your ORF of interest because some mutagenesis strategy allow the disruption of an ORF without polar effect on downstream ORFs. Do you introduce a cassette encoding an antibiotic in your construction? If you want to check the polar/non polar effect of your deletion, you can do this by comparing the transcription of genes downstream of your deletion in your mutant and in the parental strain by RT-PCR.
Yes, it may affect. Since prokaryotes have a very small and simple organization, a common promoter or intermittent promoter is many a times present of two or more adjacent / sequential genes. So, you must be careful in choosing a knock out aspect.
How can one check to rule out this possibility of common promoter for the adjacent/ sequential genes?
See. If your knock out knocks out the adjacent genes as well, then it is common promoter. Now the thing is, how to check it? Well bio-info can be of some help. I can provide you with some links. I think they will be helpful:
http://nucleix.mbu.iisc.ernet.in/prombase/about.htm
http://www.scfbio-iitd.res.in/chemgenome/chemgenomenew.jsp
Hope these helps.
Yes of course: Think of all the polar mutations in the lac- or gal-operon that you find in all Genetics textbooks.
Yes, it can affect the transcription of downstream genes if the open reading frame you are deleting is co-transcribed with others. However, it depends on how you knock-out your ORF of interest because some mutagenesis strategy allow the disruption of an ORF without polar effect on downstream ORFs. Do you introduce a cassette encoding an antibiotic in your construction? If you want to check the polar/non polar effect of your deletion, you can do this by comparing the transcription of genes downstream of your deletion in your mutant and in the parental strain by RT-PCR.
Making sure to keep the remainder of the gene - after deletion - in frame with the original gene is important for preventing polar effects caused by a frameshift in downstream co-transcribed genes. For that reason, what you say here, "After homologous recombination the knockout gene will contain 208 bp from ATG to TAA which is not in triplet frame," doesn't sound like a good idea. Keep the deletion scar in frame with the original gene if you can. There may still be polar effects if the deletion scar is in frame, so check for those like Isabelle suggested.
Thanks everyone now i m going to make new construct and will make it in triplet frame.
well, it may. because of simple reason that is prokaryotes has very less intergenic space, even some times it may hv overlap of genes.
It will depend on ORF present in bacterial mRNA . If it will be a co-transcribed genes then it will effect transcription of companion gene or near by gene in order. I may show effect upstream as well as down stream at a time.
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