No tutorial on Monday!
The miscommunication was about what we call '-': for 335 our definition of '-' will extend to the amount of expression that we get in the presence of glucose and inducer, or with a mutant crp gene.
For your exams, the -, + and ++ will be clearly defined in the question.
In general we'll say that:
mutations in the promoter, in the lacZ, in the crp gene, and lacIs mutations will give us a "-" in all cases
a WT strain grown without inducer will give us a "-"
a WT strain grown in the presence of inducer and glucose will give us a "-"
a lacI- strain grown in the absence of glucose will always give us "++"
a lacI- strain grown in the presence of glucose will give us a "-" (same as WT with inducer and glucose)
a lacOc strain in the presence of glucose will give us a "-"
a lacOc strain in the absence of glucose and presence of inducer will give us a "++"
a lacOc strain in the absence of glucose and inducer will give us a "+"
lacOc wins over lacIs....but adding inducer does not make a difference:
lacOc lacIs in the absence of glucose is always "+"
lacOclacIs in the presence of glucose is always "-"
WANT TO KNOW MORE ABOUT THE LACTOSE OPERON? Here is a recent review. If you want to see how they 'figured out' the basic idea of the lac regulatory system, check out the first reference in the review paper!
20 comments:
for eukaryotic gene, after you do southern blotting and cut out the agar plug that hybridize with your probe, you sequence it to make sure you have a complete gene. Do we need to do this sequecing for bacterial gene?
Also, after you transform the genomic library into the bacteria and you screen for the colonies containing your gene of interest. How can you isolate those particular plasmid containing your gene of interest since those colonies can take up more than one type of plasmid?
thank you very much
1. When we cut out the chunk of gel with DNA in it from a Southern blot's parallel gel, we DO NOT sequence that DNA. It's a mixture of many, many different sequences, the sequencing reaction would eb a complete mess and the result non-usable.
First, we need to clone that DNA (all the different sequences)....we make a partial library of it. Then we screen it with our probe that will recognize our gene of interest.
THEN we sequence our clone, to see if we have the entire gene.
2. It is not a matter of bacterial gene or eukaryotic gene. It is a matter of HOW we are doing the cloning. if it's based on sequence similarity (with a probe), then yes, we always have to make sure that we have the full gene. We can do this by sequencing.
However, if we are cloning through functional complementation, the fact that we get complementation tells us that we have the full gene. (And functional complementation is usually used in bacteria).
3. each colony has only one type of plasmid. Each colony is a clone of one bacterium, which picked up one particular plasmid, so all the bacteria in one colony contain the same plasmid. We screen to find which colony has the plasmid with our gene of interest. Then we isolate the plasmid from the colony so that we can use it for further experiments.
You are very welcome
Fellow student here:
Craig mentioned in class a while ago that, for the purposes of Bio 335, we assume that the bacteria we're dealing with each take in only one plasmid.
I think that both prokaryotes and eukaryotes have genes that may contain restriction sites within the genes. Thus, I think you're probably right in saying that one would need to sequence the bacterial gene after performing a Southern blot in order to ascertain that the whole gene has been obtained (just like for eukaryotes).
Correct me I'm wrong, Pam!
I agree with your answer!
Thank you for taking the time to help out your fellow 335-takers.
Cheers,
Pam
From the second (Pam's?) reply:
"First, we need to clone that DNA (all the different sequences)....we make a partial library of it. Then we screen it with our probe that will recognize our gene of interest.
THEN we sequence our clone, to see if we have the entire gene."
Doesn't this step involve another Southern blot? Then we would have to cut out the agar plug that we want and it seems that we are back at square 1 again. o_O
Thanks for any help provided!
No, no need for another Southern. You would extract and purify the DNA from your agarose chunk. Then you'd clone it into a plasmid and transform it into bacteria, thus creating a partial genomic library. Then you'd do colony lift hybridization to find your colony (or colonies) that contain the plasmid construct of interest.
The first Southern is not absolutely necessary, you just do it because it allows you to end up screening a partial library instead of a full one (and a full genomic library of a eukaryote can be very, very large....).
Cheers
Pam
What is the effect of Ic? I thought we had only discussed I-, I+, and Is?
Thnx
Did I mention Ic?
That was a typo-thanks for pointing it out, I'll fix it in a second.
But since you ask, here's the deal:
there are 2 sets of lacI nomenclatures. The nold one (which is the one used in 335 and in the book) has Is and I-.
The newer one (and only microbi lac operoners and their pupils usually learn it) calls the lacIs lacI-, and the lacI- lacIc.
It's just terminology.
Pam
both dna microarrary and northern blot study gene expression. So, do you usually use dna microarrary when you are trying to tell DIFFERENCE in gene expression and northern blot to study if dna is transcribed?
thank you
in the first southern blot before making your partial genomic library, do you add complete genomic library in each well or simply digested dna fragments?
also, can you explain why crp mutation will give us -?what if you have crp-I+ in presence of lactose, wouldn't that give you +?
thank you
hi, pam,
i am not sure if my explaination for the top question is correct. So, when it is crp-, it is as if there is a lot of glucose in the environment so it is -?
also for ch10 ques 15, for lacI- in the presence of glucose, the level of Bgal is 1 not 0. Is it because the repressor doesn't bind causing B gal to be transcribe. However, is it not a constitutive level. But if we are given larger numbers like 1, 100, 1000 can you say 100 is a constitutive level?
I'm not Pam, but I'll still try to answer the question above mine while Pam's away:
crp- means that the crp gene isn't producing the cap A protein. No cap A protein means no cap A/cAMP complex to bind to RNA polymerase to get transcription going. crp+ in the presence of high glucose means that the cap A protein is being produced, but in this case, cAMP levels are extrememly low due to the presence of high glucose, so for the purposes of Bio 335 we are not going to have the cap A/cAMP complex, either. Therefore, yes, the effect of crp- is the same as having high glucose levels in the medium.
I think that for #15 in Chapter 10, 1 is not considered constitutive. 1 here is most likely basal level (remember the analogy Craig made in class about not being able to survive on just 1% of your normal caloric intake?).
The reason we have 1 for lacI- in the presence of lactose and glucose is that the presence of lactose ensures that the repressor protein never binds to the operator, no matter which allele of I- we are dealing with (some I- mutations may produce repressor proteins that don't bind to the operator very well), and the presence of glucose results in almost no cap A/cAMP complex, so you'd only get one beta-gal being transcribed every once in a while (basal level). It's the same scenario as growing WT E. coli in the presence of lactose and glucose, and the book has already defined this level of beta-gal activity as being 1.
I'll wait for Pam to give the final A-ok on my exlanation.
Microarrays vs northern blot:
you us a northern when you are only looking at a few genes. you can use it to see if a gene is expressed in a certain tissue/under certain conditions/at a certain time, etc, or to compare the levels of expression between two tissues/conditions, etc. Microarrays do the same thing, but for all the genes in the genome at the same time.
About the crp-: it's true, having a crp- strain is like having a lot of glucose in a WT strain!
The reason why the question in the book is 1 and not 0 is because they set their "zero" at a different level (it's kind of arbitrary). I was just looking up an article on mutations in the crp gene and they say that, in the absence of glucose, a WT strain produces about 1700 units of B-gal (not sure what their units are), while a crp- strain in the same conditions produces only 50 units.
If we were to look at those results, we'd set our "-" at let's say 100 units, so anything below 100 would be scored as a "-". We may set our "++" at 1000 and our "+" at between 100 and 999.
I hope this helps
Hey 335-ers!
Good job answering each other's questions!
I perfectly agree with your explanations about the book's numbers in question 15. Remember to keep an open mind regarding those conventions (the numbers and the +s and -s): different people set their 0, their - and their +s at different levels.
We are in 335 and should follow the 335 conventions.
how do you clone a gene that is fundamental for surviving and required for all stages of development of cells (ex. actin gene) without the probe?
Thanks so much
just to clarify...there's nothing about reporter genes on the midterm right? i remember we mentioned it once in tutorial but i don't think craig talked about it at all
no reporter genes yet for you guys. And for the actin gene...it's hard. You'll need conditional mutants or hypomorph mutants and you need to have a phenotype that you can look for. Not straightforward at all. don't worry about it for now
cheers
Pam
What is rec-? and what do we need to know about rec-?
Is rec- in your notes?
If it is not, then you don't need to know about it.
It is a mutation in one of the genes involved in recombination. Lab strains are usually rec- (among other things).
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