7 replies to this topic

[tihong10]

Hi,

I'm hoping someone can explain to me how a promoterless operon works and perhaps provide an example.

I am studying an article on bacterial biofilms in which the researcher created "a library of random insertion mutants generated with a MudX transposon carrying a promoterless lacZ gene", and I want to know the behavior of a promoterless operon in order to understand why they were used in this study.

Thanks in advance.

-Ted

[phage434]

Transposons randomly insert into the genome.  If they insert in the correct oriention following a promoter, then the gene contained in the transposon will be expressed.  Otherwise, it will not.  This is a way of determining the location of promoters in the genome, and of measuring the expression level of those promoters.

[Nayeem991]

The purpose of using promoterless lacZ is to study the expression of promoter of that particular gene. If lacZ contained its own promoter it wont be expressed under the promoter of the gene which is mutated. I am making you clear with an example.... Suppose "A" is a gene which is related to bacterial biofilm formation. You want to study the expression level of gene A under some some stimuli..If you have a mutant strain where the gene is mutated with that particular transposon (transposon with promoterless lacZ) your work will be easier.  suppose u want to study the expression level of gene A when the bacteia is under nutritional deprivation. In that case this promoterless lacZ will give u a solution. you can quantitatively assay the activity of lacZ by Beta galactosidase assay. i hope u know about this assay. The beta galactosidase assay will give you the idea about the expression level  of gene A, as lacZ was expressed under the promoter of gene A.
I think its clear to you now. If you have any other query please let me know... i have practical experience of working with this kind of mutant...

Edited by Nayeem991, 13 July 2012 - 12:34 PM.

[tihong10]

Thank you Phage434 and Nayeem991 for your responses. I have a slight grasp on your explanation. Please confirm if my understanding is correct:

So, a promoterless lacZ transposon  is a unit of DNA that encodes Beta galactosidase and randomly inserts itself into the genome. The expression of the gene in which the lacZ transposon inserts itself can be measured in response to an independent variable by some marker that is proportional to beta gal concentration (e.g. more beta gal = more blue color). The transposon is promoterless in order to respond to the activity level of the promoter of the gene in which the transposon inserted itself into, and not to its own promoter.

I had two follow up questions that I hope someone can also answer:

1. How does one identify or locate the gene in which the transposon inserted itself?
2. How does one know that the expression of the gene in which the transposon was inserted is more or less than before treatment? I.E. how does one establish baseline activity of the gene in question?

Thank you in advance
Ted

[Nayeem991]

tihong10, on 19 July 2012 - 05:08 PM, said:

Thank you Phage434 and Nayeem991 for your responses. I have a slight grasp on your explanation. Please confirm if my understanding is correct:

So, a promoterless lacZ transposon  is a unit of DNA that encodes Beta galactosidase and randomly inserts itself into the genome. The expression of the gene in which the lacZ transposon inserts itself can be measured in response to an independent variable by some marker that is proportional to beta gal concentration (e.g. more beta gal = more blue color). The transposon is promoterless in order to respond to the activity level of the promoter of the gene in which the transposon inserted itself into, and not to its own promoter.

I had two follow up questions that I hope someone can also answer:

1. How does one identify or locate the gene in which the transposon inserted itself?
2. How does one know that the expression of the gene in which the transposon was inserted is more or less than before treatment? I.E. how does one establish baseline activity of the gene in question?

Thank you in advance
Ted

your understanding is correct. Actually commercial kit is available to assay beta galactosidase activity and it is expressed as "Miller Unit" after some mathmatical calculation. You can google it for better understanding.   The gene in which the transposon is inserted is identified by two round of semiarbitrary PCR followed by sequencing. The junction sequence is first identified and then the gene is identified using some bioinformatics tools. For better understanding you can read this paper.... http://www.pnas.org/...05/25/8736.full I hope it will help you for better understanding.About your second question I would like to say that, in every biological experiment control is an essential part. If you want to determine the effect of a particular thing on expression of your gene of interest, you need to use a control where you wont add that particular thing. Suppose you want to assay the expression level of a particular gene in response to increased salt concentration. In that case you  will fix the baseline of expression at normal salt concentration then you will compare that with increased one. Is it clear to you now?

[tihong10]

Hi Nayeem991,

I understand your explanation of the first question; however, I think my second question wasn't worded adequately to express my confusion. If the experimenter doesn't know which gene the transposon will insert itself into; how does he or she know which gene to get a baseline reading of? Does the researcher first identify which gene was mutated then take a baseline measurement of that gene?

Thanks,
Ted

[Nayeem991]

tihong10, on 21 July 2012 - 10:51 AM, said:

Hi Nayeem991,

I understand your explanation of the first question; however, I think my second question wasn't worded adequately to express my confusion. If the experimenter doesn't know which gene the transposon will insert itself into; how does he or she know which gene to get a baseline reading of? Does the researcher first identify which gene was mutated then take a baseline measurement of that gene?

Thanks,
Ted

Actually the baseline reading is not a constant thing. whenever you perform an experiment you should take 2 sets of samples. One with the test material and another is without test material which works as control. Then you have to compare them to get an idea about the effect of your test material on expression of the particular gene. Suppose you have 4 mutants A, B, C and D (you don't know where the transposon has inserted)and you want to study the effect of increased salt concentration on these 4 mutants. For this experiment you have to take two sets of sample. Set 1 would be with normal salt concentration and set 2 would be with increased salt concentration. Then comparing the result of set 2 with set 1 you can identify which mutant is affected by increased salt concentration (expression level may increase of decrease). You may take multiple copies of both sets for better comparison of your result. Suppose expression level of B is increased due to increased salt concentration, then you can identify what actually mutant B is (Where the transposon has inserted). So, identification of insertion site is not important for initial screening. But the fact is, this kind of transposon is generally used to create a mutant library. In that case you  have to identify the insertion site first. Because to create a library you need to have mutant of every possible genes of that organism (except the genes responsible for the survival of that organism). If you read the paper (which i recommended) you would be able to understand how the mutant library is created.  Is it clear to you now? please let me know if there is any further query....

[tihong10]

Thank you Nayeem991 for your detailed response; I have a better understanding of how these transposons are used and will refer to the article for more information