Question on Transcriptional Fusion lacZ - Question on Protocol (Jul/20/2006 )
I know this might be a simple procedure for others, but im really at lost coz i dont know how to make a protocol for this, i havent done this before. The case is like this, I was told to clone the promoter region of gene X in Salmonella typhimurium 14028s and measure promoter activity/induction in stationary phase. Well my understanding (based on journal, book readings) is i can do this by lacZ transcription operon fusion (from a promotorless lacZ plasmid) with the promoter from my gene x in Salmonella. The activity will be measured using beta galactosidase assay. Is that right?can somebody pls explain this to me?I would really appreciate it. I dnt know which plasmid to choose and how to do it.Im really lost. If you can give me reference for this and the protocol that would be a very big help.Thank you.
-arvinsign-
QUOTE (arvinsign @ Jul 20 2006, 09:32 AM)
I know this might be a simple procedure for others, but im really at lost coz i dont know how to make a protocol for this, i havent done this before. The case is like this, I was told to clone the promoter region of gene X in Salmonella typhimurium 14028s and measure promoter activity/induction in stationary phase. Well my understanding (based on journal, book readings) is i can do this by lacZ transcription operon fusion (from a promotorless lacZ plasmid) with the promoter from my gene x in Salmonella. The activity will be measured using beta galactosidase assay. Is that right?can somebody pls explain this to me?I would really appreciate it. I dnt know which plasmid to choose and how to do it.Im really lost. If you can give me reference for this and the protocol that would be a very big help.Thank you.
u will be doing reporter gene assay then as far as i understand.
First of all, u should decide on which reporter gene u will utilise. i knew few of them and each provides you with different facilities and the choice of reporter gene depends on your conditions indeed. your reporter may be lac Z but doesn't have to be so. u may also use luciferase, CAT, SEAP as reporter gene. u should consider the detection method you will use during your reporter assay. how will you measure the promoter activity (how much is your reporter gene expressed under the control of that promoter region?) for example, for luciferase assay you need to have a luminometer; CAT assay depends on radioactivity; SEAP is an alkaline phosphatase which gives a yellow color when u incubate it with its substrate so you can do spectrophotometric measurements. SEAP is also a secreted protein so when you transfect your cells with your reporter assay plasmid, you will not need to purify the protein in order to measure the activity of your promoter. SEAP is just secreted so you can simply take the medium and do your measurements. all of those reporter genes are found in commercially available vectors.
there are more of those reporter assays which i don't know in detail. you may google it as reporter assay/reporter gene assay.
i hope this will help;)
-dodosko-
QUOTE (dodosko @ Jul 20 2006, 10:14 PM)
QUOTE (arvinsign @ Jul 20 2006, 09:32 AM)
I know this might be a simple procedure for others, but im really at lost coz i dont know how to make a protocol for this, i havent done this before. The case is like this, I was told to clone the promoter region of gene X in Salmonella typhimurium 14028s and measure promoter activity/induction in stationary phase. Well my understanding (based on journal, book readings) is i can do this by lacZ transcription operon fusion (from a promotorless lacZ plasmid) with the promoter from my gene x in Salmonella. The activity will be measured using beta galactosidase assay. Is that right?can somebody pls explain this to me?I would really appreciate it. I dnt know which plasmid to choose and how to do it.Im really lost. If you can give me reference for this and the protocol that would be a very big help.Thank you.
u will be doing reporter gene assay then as far as i understand.
First of all, u should decide on which reporter gene u will utilise. i knew few of them and each provides you with different facilities and the choice of reporter gene depends on your conditions indeed. your reporter may be lac Z but doesn't have to be so. u may also use luciferase, CAT, SEAP as reporter gene. u should consider the detection method you will use during your reporter assay. how will you measure the promoter activity (how much is your reporter gene expressed under the control of that promoter region?) for example, for luciferase assay you need to have a luminometer; CAT assay depends on radioactivity; SEAP is an alkaline phosphatase which gives a yellow color when u incubate it with its substrate so you can do spectrophotometric measurements. SEAP is also a secreted protein so when you transfect your cells with your reporter assay plasmid, you will not need to purify the protein in order to measure the activity of your promoter. SEAP is just secreted so you can simply take the medium and do your measurements. all of those reporter genes are found in commercially available vectors.
there are more of those reporter assays which i don't know in detail. you may google it as reporter assay/reporter gene assay.
i hope this will help;)
Thank you dodosko for the reply. Actually youre right. Im supposed to do a reporter gene assay using lacZ. I know thr are methods but i might be using this one. and this is my problem, i dnt know how am i gonna do it.
-arvinsign-
oh, i see 
then u may want to check this websites:
http://www.biocompare.com/matrix/3854/Andb...ase-Assays.html
http://www.clontech.com/AIT/Ecommerce/Clon...8|160|71|133|42
in the latter site, u'll find a list of products. the first one may fit you.
-dodosko-