EXTRA HELP SESSION(S)
I'll be in the help office on Thursday from 4 to 6 pm. I could be persuaded to do some extra help sessions on friday morning (I have a workshop 2-6pm) or sometime during the weekend for about 2 hours.
You people decide (deadline: Wed. at 10pm)!
Pam
posted by pam39 @ 11:43 AM
33 Comments:
Hi Pam,
Can we meet up on Friday at around 12:30ish? Where are we meeting?
Thanks,
Ada
Friday doesn't work for me since i have class from 8-2pm. Weekend works best.
For those who were in today's (tues, Jan 30) tutorial, I have sent a total of 8emails with all the practice problems Pam gave us today. If I'm missing any problems please email them to me and I will forward it to everyone else. If there was a Justin in today's tutorial, I wasn't sure if I got your email correctly so if you see this, please post up your email here. I will check here at least once everyday. Thanks a bunch everyone.
Belinda
Thank you for you time Belinda!
Hey,
I was review my notes again and was wondering you anyone can help me clarify this, it's from Lecture 4 page 5, the 3rd genotpye with the F plasmid added. I do not really understand how that is acting in cis.
Appreciate your time.
Hi Belinda,
I was very sick today (FLU...) and couldn't come to the tutorial. I was wondering if you could send me the problems too. My name is Iran and my email is: t_iren7@yahoo.com
Thanks alot,
Iran
Hi guys,
I'm refering to Lecture 5 page 2, bullet II...it says "Because the probabilty of inducer-binding domain-specific mutations is less than the combined probability of loss of DNA-binding and ot all protein functions, most repressor mutations result in constitutive transcription of regulated genes." Does this statement mean that even when there's a mutation in the inducer-binding site, proteins would still be made, except in very low amounts instead of not being made at all? What are we suppose to say on a test then?
Can someone help?
Thanks!!!!!!!
Belinda, thanks for helping everybody out!
Sorry for interfering in your discussion....
The mutations business with the lac repressor:
1- the protein can be made in normal amounts, but with a mutation in the inducer binding site mutated, so it never falls off the operator.
2- i like to think of the whole concept this way:
the job of the lac repressor is to repress. lacI- is a mutant that does not do this job (or does it less well), lacI[s] does its repressing job too well. Now, let's think of a brand new, well working bike. if we throw it out the window (from the 11th floor), three things can happen.
1) the bike gets damaged and does not work as well anymore (most likely outcome).
2) nothing (not likely)
3) the bike will work better after this 'treatment' (very unlikely).
Same thing if you randomly mutagenize bacteria...it's much more likely to get mutants that work less well (lacI-) than mutants that work better than WT (lacI[s])! We can get lacI- by mutating the lacI promoter, by mutating the DNA-binding domain, by having a nonsense mutation that result in truncated protein, etc etc...but there are very few specific substitutions that can result in mutations that specifically make the lacI not bind to the inducer.
Glad to see you guys are discussing!
Pam
Hi Belinda, is it possible to send me the problems? Because the ones in pam's box are gone! my email is wiz4017@yahoo.com
Thanks.
Victoria
Hi Belinda, can't find the problems in Pam's box. Can you send them over? arennj@interchange.ubc.ca
Thanks,
Arenn
Hey Belinda,
I missed the last tutorial and would love to get a copy of those questions.
Could you send them to DerekLMartin@Shaw.ca
Thanks
Derek
hi Pam, is there any way that you could possibly expalin question 1 on page two of the 2006 12:00 section midterm exam? it's a tough one to understand. Thanks
it's nice to know that I'm not the only one who feels lost with this question, in thoery it should be doable, but it's difficult
I think it's from the 2005 exam no? yes..I find that question difficult to understand as well because how are we suppose to know that there is some kind of a cross over happening?
no, the question above is similar to the lac operon stuff, it had a bla gene and a tet resistant site on it and you have to fill in a table.
it's confusing when, is the bla gene a part of the lac operon only and the tet site is not? or are they both a part of the lac operon? just have no idea what to do
Bla gene, here we come:
the bla gene is a gene that makes cells resistants to ampicillin. You clone it into a plasmid downstream of an 'artificial' lac promoter+lac operator. The plasmid also contains a gene that confers resistance to tetracycline.
What you have to remember is just that the cells that received this plasmid will be always tetracycline resistant, and amp resistant only in the presence of IPTG or lactose. it also says that the cells you use are met- (and they will always be met- regardless of whether they have the plasmid or not).
Hope this helps.
(You guys will be fine, don't panic!)
Cheers
Pam
what part of the question says that it will be AMP resistant only when lactose or IPTG are present?
so when you have met + tet + amp on the minimal media, will there be coloby growth from the tet risistant part of the gene? if not then why not?
I see the dilemma.
The question says that the bla gene on the plasmid has a 'lac promoter' (the same promoter that usually drives the lacZ. On the map it says lac PO (lac promotor and operator), and since you introduce this plasmif in met-, but otherwise WT cells, there is lacI repressor produced-which will block the expression of both the endogenous lacZ and the 'artificial' lac PO-bla gene construct.
On MM+met+tet+amp you won't get colonies, because the bla gene can't be expressed (since it's repressed by the lac repressor) so the ampicillin will kill the cells.
Try to always think about it from a molecular point of view, one gene at the time...e.g. is the tet resistance expressed? yes, because it is just the regular tet gene, and does not need an inducer. Is the amp gene expressed? In this case its promoter is inducible...so it's only expressed when the inducer is there. Do we have inducer in the medium?...
Remember that WT bacteria are sensitive to all antibiotics!
Have a good night and get some sleep this weekend-brains need to rest in order to function well!
pam
what about when you just have
MM + met + tet? will you have colonies?
even though the tet gene is present in the chromosome, it won't be expressed into colonies? is that what we are to assume for the question above and for the
MM + met + tet + amp as well (that's what there is no colony growth)?
Pam please help with this question, it seems like I have missed the whole point. bla is part of the lac operon, but tet is not? but will there be growth with the tet present in minimal media? I really need this in baby steps Pam, please, please, please :)
For the person who posted at 10:16pm:
MM, met, tet and amp wont grow any colonies b/c ultimately you do not have IPTG to have any inducing for the bla gene. If the bla gene is not made, then it means that it can't be amp resistant. Just like with strain 8, colonies will not grow b/c you do not have IPTG to make sure that bla can be amp r.
I think when you have jsut met and tet on the plate, it will grow b/c your plasmid is tet res.
Hope that makes sense. If Pam can confirm this or correct me, it would be great.
this is my answers to the question:
1) no n/a
since it is met- it will not grow
2) yes white
met and tet are present so colony will gorw since we have tetR
3) no n/a
no colony gorwth because no inducer present to start the lac operon system
4) yes white
inducer present and lac operon system works fine, but only tetR colony gorwth observed since no amp on media
5) yes blue
we are told that everything is wild type, so X-gal will cleave B-galactosidase since we have Z+ gene we will have blue colonies
6) no n/a
no inducer present
7) no n/a
missing tet on media, so no gorwth
8) no n/a
no inducer or tet present
I am not sure if this is correct, please lets discuss and figure this out before the dreadful midterm tomorrow
something doesn't feel right about those answers, especially number 5
Hello to the one who posted the answers up:
My answers for 5 and 7 are different from yours but the rest are the same!
For 5, I thought it would grow but it would have white colonies b/c if the medium does not have tet in it, then it's allows for both tet r and tet s to grow. And it's white b/c you do not have IPTG to make the Z gene which then cleaves the X gal.
Because you dont have tet in the medium so I dont think you need to worry about it .
Let me know if there are flaws.
Hi,
I forgot to check this post the last two days so I didn't see all the people requesting questions. So sorry. I'll send them right now even though it seems pretty late. Sorry again.
Belinda
so, we don't have to worry whether or not tet is present in the minimal media, there will still be colony gorwth?
Derek,
I just sent the practice problems out again and I got an email back saying it couldn't be delivered to your email. If you still want the questions please email me back at belindalai666@hotmail.com
About the amp and the bla gene: the person who posted at 12.30AM is correct.
Step by step: you ALWAYS express the tet resistance, so colonies will grow on all media that have no antibiotics or tet only.
You are ALWAYS met-, so you only grow on media that are supplemented with met.
Whenever you have inducer (IPTG) in the medium, you express two things: the lacZ from the bacterial genome, and our 'artificial' bla gene from the plasmid. Whenever the bla gene is expressed, the cells become amp resistant.
So: no met--> always no colonies
met+tet--> always colonies (white)
met--> always white colonies
met+tet+amp---> no colonies (the amp kills them off)
met+amp+IPTG-->white colonies (IPTG allows expression of bla, which encodes amp resistance)
met+tet+amp+IPTG--> same
met+IPTG+tet+X-gal-->blue colonies
met+IPTG+X-gal+tet+amp--> blue colonies
met+X-gal+tet--> no colonies
The 'purpose' of the tet is to kill off all bacteria that do not have a plasmid. So without tet you get growth of cells that have plasmid and also of those that don't.
Cheers!
Pam
met+X-gal+tet would yeils colonies wouldn't it? Since met and tet are present we will still see white colonies right?
yes..that is what I think too...but the answer says otherwise..are we wrong?
Post a Comment
<< Home