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how to determine a plant promoter activity in bacteria


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#1 mbsea

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Posted 20 August 2011 - 06:06 AM

Hi everybody,

i am studying on a plant gene promoter. i identify its sequence but i want to determine its activity. I can' t do it by plant cell culture in my lab so i must try it by bacteria. Does anybody know or offer a protochol or a kit for this?I want to clone this promoter and determine its activity in bacteria by calculating luminometer. I am waiting for your advices.
Best wishes...

#2 phage434

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Posted 20 August 2011 - 06:19 AM

Plant transcription and bacterial transcription differ so greatly that any measurement you make will be meaningless. The signals needed to transcribe plant genes are very different and much more complex than those used by bacteria. In general, any transcription from a plant promoter in bacteria would be accidental, and not related to the activity of a promoter in its natural context.

#3 mbsea

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Posted 21 August 2011 - 04:47 AM

Thank you very much for your answer. You mean that i have no chance with it. How dou you think i can determine that the sequence i found is a real promoter? Could you please give me an advice about that?
Best wishes...

#4 phage434

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Posted 21 August 2011 - 06:17 AM

Your best approach would be to fuse the promoter to a reporter gene active in the plant of interest, then transfect your plant with that construct. Not easy, and certainly not the first thing you should attempt.

#5 mbsea

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Posted 21 August 2011 - 07:02 AM

thank you very much again for your answer. but this is a bad news for me. because i was expecting to identify the promoter activity by using bacteria. anyway, could you please offer me another topic for a promoter. what could i do with a promoter sequence? i need to design another experiment because i can't do a transgenic plant in my lab.
best regards to everyone...

#6 mbsea

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Posted 04 September 2011 - 11:37 PM

if my question is so silly sorry about that but my supervisor says that we can look for this by using yeast. Is there any luciferase assay kit for this application? i searched but couldn't find. thanks...

#7 phage434

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Posted 05 September 2011 - 06:54 AM

Yeast transcription is much closer to plant transcription (they are both eukaryotes). So, the experiment might have some meaning. Still, there are hundreds or thousands of transcription factors which will influence transcription in a plant which will be missing in yeast. This experiment makes some sense, however.

#8 mbsea

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Posted 08 September 2011 - 01:51 AM

do you know any kit for this?

#9 mbsea

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Posted 20 November 2011 - 12:29 AM

Hi,

did anyone try clontech pmetluc reporter for plant promoter activity test in yeast? or do you know if it is posssible to do?

#10 Evanescence

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Posted 24 November 2011 - 10:27 PM

Phage was right as he knows and experienced many things in this field, i guess you should tell us what you want to do... You can try GFP plasmid...luciferase was fine too...but i gonna to ask, if you try on yeast then how yu can convince people that it workable in plant..sometimes it cant cooperate well each other..for me, you still need to transfect the plant to conclude your experiment..i dont know though just sharing my opinion about this...by the way you can check the promoter prediction at softberry.com and like phage told you...there are big different between plant and microbes promoter..anyways i wish you all the best thing ..dont give up on anything :) _evanescence

#11 mbsea

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Posted 29 November 2011 - 05:27 AM

Yes, i agree with you.The thing , want to do is: I have a plant beta glucosidase gene promoter sequence. I want to test if it gives promoter activity. But i can't test it with transgenic plants in my lab.So, I want to use yeast expression system if possible. Despite all my search, i could't find a kit proper for this. Isn't there any study before like mine? I couldn't find it either:(

#12 Evanescence

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Posted 29 November 2011 - 05:44 AM

HI :)
Hmm first maybe you should find the system for yeast and do transfection once you got one...but based on what i know, normally they already contained promoter in the plasmid...otherwise you need to get the backbone that suit for yeast transformation and construct all by yourself...it will takes time, try to search transformation in yeast let see what you will get...I guess transformation in yeast is not rare, so you will have lot of method just in different strategy..Do you really need to use that promoter? i mean if you cant try it directly on plant it's hard to know the effectiveness of the pomoter right? maybe if you can do transgenic plant, agrobacterium can help you :) but for me yeast can work too...as long as you can get suitable system for (plasmid) once you insert Promoter+Gene then just use electroporation ...:) hope it works..

#13 mbsea

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Posted 29 November 2011 - 08:03 AM

Thank you very much. I hope i can carry out this.




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