Blog

During the summer the students and some of the buddies have blogged about their frustrations with their experiments but also about their SUCCESS!!! The sporadic blogs follow...

August 15, 2008 8:38 PM

by Alex Ng

team silencing has retired....goodbye people, i will probably be dropping in whenever i'm in the city, good luck, and jimmy, dont screw up our experiments. I will see you guys whenever i come back.

1 comment

August 14, 2008 10:52 PM

by Jimmy Huang

I actually beat my previous year's record of working!!!

It is 10:52pm!!

I have beaten my last year's record of working till 10:42pm!!

The IGEM people are the only people here!!! We own the lab!!!

~Jimmy

0 comments

August 5, 2008 8:48 PM

by Tiffany Saw

So its one of those days again.

Things have been going quite topsy-turvy lately.

Nili, Cathy, and Willis all left us previously and now Xili, David, and Jacob will be leaving us, too - what an empty lab!

As for the actual lab work, wellllll, you could look at it one of two ways:

On the one hand, anti-silencing seems to finally have a promising candidate, Esa1. On the other, the oscillator is facing some issues at the moment, and will not be resolved until I get my sequences tomorrow (gah, I want those sequences).

So basically, it's one of those days where I just sit around and watch Jacinto do lab work while I eat. Just kidding. I've been doing some little chores that will help both in due time (which is pretty soon, actually, whoo).

As for now, I've ticked off everything on my to-do list (until I get my sequences, that is) and am just waiting for Jacinto to figure out how to use that marker pen.

-T. Saw

1 comment

August 4, 2008 10:52 PM

by Jimmy Huang

To Alex & Noah

Noah Noah Noah Noah Noah!!!

That riddle you gave me concerning how to integrate a single copy plasmid into yeast and how we prevent multiple copies from going into the same yeast!!! I think I understand / have an answer to that riddle. Although it's like 2ish in the morning, I was just thinking about stuff and it came to me.

It has to do with the selective media + the place where we linearize our Plasmid!

So usually, when we integrate plasmids into yeast, we linearize it through cutting it with an enzyme. And the cut we usually do resides in a Selective Marker.

You said yeast love to do homologous recombination, and as such, they sometimes integrate multiple copies of plasmids at one site. Sometimes that's okay, but other times we don't want that.

So your question was, how do we get a single plasmid integration rather than multiple copies!

So I was thinking about that hint, 'homologous recombination.'

When we cut our plasmid, the selectable marker is broken. And this is key because the broken marker is taken into yeast through homologous recombination. When the yeast does that, they no longer have the functional gene, ie: LUE

So when we transform yeast, we plate on -LUE or -Selectable Marker because this will allow us to screen for yeast without that functional gene.

The reason why we can screen for a single plasmid integrant is because yeast that integrate our plasmid multiple times will have a functional selectable marker, which won't grow on our selective plates! Or maybe they do... I'm not sure exactly, but hopefully this is close to the right answer~ If not, I'll have to think some more in my random times of random thoughts...

So yes... this is my answer/guess to your riddle. Tell me if I'm right or wrong Monday =)

~Jimmy

8/4/08 : 10:42PM = UPDATE

I am wrong. =(

But that will not stop mee! I will continue to try and solve this riddle! I have a feeling I might've not been too clear, but to clarify.

Yeast have 'broken' genes such as; LUE, HIS, etc. Which is why we grow SF992 on YPD, which is a rich media that has all of these components yeast require. When we integrate our plasmids into yeast, we cut in the working selectable marker which will undergo homologous recombination in yeast. When yeast integrates our copy of the functional marker into their genome, they can then survive on minus 'media' plates because they can produce it.

Thanks Andrew for the refresher =) It made me realize I wasn't being clear in my writing what I was trying to convey.

Don't worry Noah, I did not ask Andrew for the answer. I will try to solve this myself, or with Alex =P Since you asked him too. Also, thanks for reminding me, visually, how they recombine. Through the drawings I understand how they can integrate multiple copies of our plasmid which also allow these yeast to grow on our minus 'media' plates.

So the question still remains: "How can we modify our components so that there will only be One integration rather than multiples?"

~Jimmy

0 comments

July 31, 2008 6:07 PM

by Tiffany Saw

Quite...

At the moment, I'm quite confused because everytime I look at the clock, I swear it's going backwards, which makes me wonder, "When the hell is this digest going to be done?" To my dismay, time is NOT running backwards (that would've been awfully useful) and I'm just being supremely impatient and anxious for my test digest to finish so I can either proceed or go back to step one - mini-preps (not that I'm complaining; I love mini-preps!).

I'm also quite hungry. I gave David and Lingli my Cheez-Its and fruit and now realize that I only have chocolate and cheez-its left in my bag of endless food. Thank god I have a secret stash of 100 Calorie Bars hidden around here - but I digress.

So I've been working on quite a few things lately: AAR1 digesting like crazy for Leeza's mass ligations/tranformations, ligating pFIG into pRS303 (everytime I think I got it, a new problem comes up - you've got to be kidding me!), analyzing FACs/FLOJO data, THE HORRIBLE COLLEGE APPLICATIONS AND THE NIGHTMARE-INDUCING AP HOMEWORK, and recently, my new "project" which I'm quite excited about - the oscillator.

Okay, first off: What the heck is an oscillator? Well, according to thefreedictionary.com, the physics definition is "to vary between alternate extremes, usually within a definable period of time." Think of Peking's project last year and their "oscillations" between the various parts - get it, yeah? yeah? yeah? Okay, good.

Second off: Why am I so excited? Well EXCUSE ME FOR BEING A NERD, JEEZ.

Other than that, there's not much lab-related stuff to talk about, but just because I'm still bitter about it (and even though you all have probably heard this a million times by now) I will say it again: I CAN'T BELIEVE I FAILED MY DRIVING TEST BECAUSE I DRIVE TOO SLOW. WTHECK. HOW DOES THAT WORK? The lady just didn't like me, I'm telling you! >=(

Oh well. I'm over it (not), but I have to weigh out the agarose powder and whatnot now, so until the next time: happy gelling :]

-T. Saw

[That was quite a long blog...hahaha]

2 comments

July 31, 2008 11:45 AM

Filamentous growth?

Noah borrowed the Gal80 knockout strain Alex made before for his own experiments and somehow the yeast were growing weird. filamentous perhaps? so we checked the cells under the microscope and it looks very filamentous indeed. need to investigate further and solve the mystery. any pictures of true yeast filamentous growth would be much appreciated for comparison of success. It seems Noah has perhaps joined the outputs group indirectly.

[The Winner's Club]

0 comments

July 30, 2008 5:42 PM

JacCat Success

So this week at the El-Samad lab, team death has encountered much failure.... NOOOOT! In fact, we have had much "good shit" of late. Jacinto and I have been working tediously to construct plasmids that will get yeast to filament. Now I don't want to get into specifics, because I don't want to spoil our progress meeting news for Friday. So I'll just say we are quite happy with our results. There will be pretty pictures for progress meeting attendants on Friday. It's not an ultra-mega-supreme-110% ideal result, BUT it's close enough to deserve a "SUCCESS!" Furthermore, we are quite pleased with this week's progress. Just yesterday, I ran a gorgeous 32-lane agarose gel. The amount of plasmid was so... massive... that an ethidium-bromide colored line could be seen across the gel. Ah, I love stuff like that. Almost as much as I love... labeling. ALMOST. My cocoa is quite stirred, indeed. Anyway, Lingli and I will hopefully be able to acquire results on alpha-factor secretion [and eventually, antimicrobial peptide secretion!] in yeast by next week. I will not be here on Friday [frown] to witness the response of awe from the progress meeting audience at the sound of team death's news, so I'm just doing my part in the updating. However, Jacinto will be left to endure in the glorious moment. Oh, I wish I could attend the protosilencers discussion! Somebody please record? Kekeke. Well, labwork beckons! I hope you all are having a good week. Til then... happy pipetting!

Cathy

2 comments

July 28, 2008 8:53 PM

by Jimmy Huang

Huang & Ng Lab

The Lab is Oursssss!

There's No One Here!

Reid just left like five minutes ago and me and Jimmy realized that EVERYONE in the lab is gone. WE HAVE TAKEN OVER!!! we have taken over the music and are now playing the music we actually want to listen to...This is AWESOME!! So if anyone wants to join the Huang and Ng lab come over RIGHT NOW. This is a limited time offer. this needs to happen more often.... Remember, LIMITED Time OFFER!~

~Alex + Jimmy

2 comments

July 27, 2008 3:48 AM

by Jimmy Huang

Anyone running FACs Monday?

So uhm, Andrej's helping me set up cultures Sunday Night, (thanks Andrej), but I realized I didn't book a FACs time.

I think I heard someone say that they had FACs on Monday, so I'm wondering if I can bum 8 wells offa' your run. Just let me know via this blog, facebook, aim, telling me Monday morning, or calling me.

But if I heard wrong, it's no big deal. I can run with Alex & Ryan Tuesday. Thanks guys.

I just thought about this while taking a shower... Haha, it's been a long day.. It's already 4am almost X_x;;

~Jimmy

0 comments

July 18, 2008 6:50 PM

by Andrej Ondracka

Hi everyone

So, I should probably also say something on the blog. I'm totally out of ideas, because I have absolutely no experience with writing a blog... I could discuss my experiments, but you just heard about them earlier today at the meeting... So basically, I am just gonna say that I'm enjoying my summer in San Francisco a lot. Thanks to all you guys, I m having a terrific time here in the lab, it's really nice to work with you all!

Andrej

1 comment

July 17, 2008 9:38 PM

by Jimmy Huang

The Success, The Preliminary Success and the Post-Poned Success.

As we all should know, if the word Success is in the title or mentioned anywhere within the first 3 words of a sentence or phrase, it is highly likely the author or person speaking is Jimmy.

It's late. Andrew has left because he has a farewell party to attend. I wonder if he was late, again. Haha. I'd hope not. I've finished most of my work for the night, but I'm still thinking about how to do my "Double Digest Test Orientation of Preliminary Success."

Most of us know or if not everyone must've heard me say it at some point, but I have three different projects. All of which don't quite fit together, but they also don't deviate far from the topic, Epigenetics.

I won't go too in-depth in each project as I will probably explain them at some point, but they're all moving along at the same speed, some a day or two ahead, and some a day or two behind.

The Success My priority project that's up on the wiki is almost near completion as I finally got a new batch of Elongase to make my PCR product which allowed me to ligate it into the cassette I've prepared almost two weeks ago now, so I just transformed my 'cassette' into TOPO ~2 hours ago, so once I send those in for sequencing tomorrow, I will find out if I have my finished product that I can transform into yeast and test the theory of "Regional Silence." I bet you've seen my beautiful advertisement for that project ;)

The Preliminary Success My third project is still a secret as even I do not fully understand what exactly we are trying to do with it, but the 'cassette' for this project has been created. But since it's a single cut vector with one site, the fragment I'm trying to ligate in has two orientations it could ligate into. I am currently trying to devise a plan of how to tell which way my fragment has ligated itself. Hopefully one of the six has worked, because if one of them are successful, it means this project will move out of the 'preliminary' success, and into the Total Success section ;)

The Post-Poned Success My second project that has to deal with Andrew's project with the BAH's is ready to under-go ligations, but I haven't had a chance to do them because of the other two projects. You might be wondering why the second project is post-poned whereas the third project is pushed forward. The reason for that is because the first and second project are too alike one another, and I didn't want to confuse myself, thus the post-ponement. So why is the second project in "Post-Poned" Success? Well, I have all the fragments and proper vectors prepared, so once I do a simple gel purification and ligation, they should be ready to xform for Success! Hehe.

Alright, back to work. I must've spent the last 15 minutes reading, commenting and creating blogs. I blame Ryan and Alex, because they walked over here and told me to look at Cathy's failures, which then led me to seeing Ryan's blog of success, which in turn instigated me into creating my own blog of success =] Must. Devise. Plan. For. Success!

EDIT:

9:35:26 Post Meridiem.

I HAVE FOUND MY PLAN FOR SUCCESS!!!

AGE1 + XMA1 <3 Success = ~ask andrew for Left Flank cut with Xma1 + 150 + 250 + 700 + 450 = Success Failure = Linearization or < than the above piece :(

~Jimmy

0 comments

July 17, 2008 8:53 PM

by Ryan Quan

'''The good, the bad. and the successful'''

so the good news is that me and alex are getting some good data from the FACs we are running. all the data is leading us to more experiments which also qualifies as good news too. The bad news is that we had a mix up with vectors. Andrew has two different vectors for Cyc1p and Adh1p. He has a: 315 Cyc1p A 315 Cyc1p A! 315 Adh1p A 315 Adh1p A! the forget what the difference is between them but something about a deletion of an amino acid. so the bad news of this is we didint know which vector we've been using because the tube we have are just labeled "315 pCyc1" and "315 pAdh1." To try and solve this, alex and i test digested each vector and ran them along side its corresponding uncut plasmid. According to our gel, something may be wrong wiht andrew stock "315 Cyc1p A!" To try and solve this, we are sending all the vectors in for sequencing. This may sound meaningless to you guys but if you got 315 pcyc1 or 315 padh1 vector from me or alex (i know jacinto did) we are not sure which vector it is. some more bad news is sir4 is being a quite difficult to get into the 315 cyc vector. i must have tried ligating sir4 3 times and gel purifying it 2 or 3 different times. im going to send in my sir3 and sir4 plasmids in 315 pcyc and 315 padh1 for sequencing also. now on to the success. me and alex are testing sir with mcherry in the expressions vectors also so we xformed them yesterday and it worked magnificiantly! (spelling?). more facs for us. also the data from sir3 in 315 vectors and ssn8 in 315 vectors looks like it did improve silencing so thats a success too. thats all for now.

1 comment

July 17, 2008 5:53 PM

by Cathy Lu

No Good Shit

First of all, permit me to justify my blog entry title. I don't know if all of you know this but in Hana's Lab, I always verbally express my approval of something via "Good Shit." I'm assuming by now, everyone has noticed that I fancy this catchy phrase. Tiff, Leeza, and Jacinto have definitely heard this come out of my mouth mutliple times. However, this week has displayed NO GOOD SHIT whatsoever. It's like when nightmares repeat night after night. In this case, I'm referring to transformations for ligations. Is it just me, or are none of the transformations working? I'm pretty sure it's the latter one, because Jacinto has not been getting results from transformations either. The plates aren't the problem, says Jacinto who consulted Andrew. The cells shoudn't be either, because I made a shopping trip over to the Lim Lab for the sole purpose of using Angi's ever-popular (very) competent cells (at least I think I recall David giving cell-prep credit to Angi...). Maybe it's the ligase buffer? Or! Maybe it's just our lab. We'll just have to wait a see, because Willis has just agreed to transform the ADH and CYC plasmids Lingli and I have been having trouble transforming (as if we haven't had enough trouble transforming everything else). This ordeal is leaving me taskless. I have to resort to wiki blogging. Kidding! Speaking of tasks, I believe I have a transformation to tend to. This whole thing makes me sound pessimistic, doesn't it? ...No, I actually think this whole blogging about our massive failure just makes us sound like noobs. And we are definitely not noobs. On a lighter note, WE HAVE THE BEST MUSIC IN OUR LAB. Good shit (or lack thereof, regarding transformations). Til then, I hope you guys are receiving successful transformation results. Cathy, blogging on behalf of Hana's Lab. By the way, I don't normally use a large number of swear words in person... (unless I'm upset, and I currently am not) And if this blog is inappropriate, I don't mind if it gets deleted. We've been told to blog, so we did so.

7 comments

July 10, 2008 12:07 PM

by Sommovilla, Nili

Jimmy Ignored me allll last summer...

Nili here. Not the "Nili Sommovilla" that seems to have multiple personality disorder, but the real Nili. I am excited that Jimmy seems to realize how easy the wiki can be. I have two responses:

1. JIMMY! I told y'all EVERY day last summer that the Wiki and Documentation both contribute to the scoring at the Jamboree! Why do you think I was always annoying you guys about it? *sigh*

2. One glitch in this--this wiki is actually for us to track our progress through the summer, and differs from the wiki we have to make for the iGEM folks. This has to be completed around a week before the Jamboree (it is "Frozen", and has to contain all the relevant info to our project in an easy to understand format. Do you even know what our wiki ended up looking like last year? You should check it out at : http://2007.igem.org/UCSF . We should all do our best to help with the wiki and documentation. It's a lot of work for only one person or two people to do, but if we all chip in a little work, it'll get done really easily.

Ok, that's it for now......

~Nili

0 comments

July 10, 2008 2:03 AM

by Jimmy Huang

Andrew Beat Me To The First Blog?!

In response to Andrew's first Blog: I am most impressed by the quickness of your blogging Andrew.

So your question/concern was that silencing through the Yeast Mating Pathway is a transient event, lasting only for a short while. What if we create a circuit, a positive feedback-loop, that amplifies the silencing induced by stimulating with Galactose? What I'm trying to say is that we should try to build something that tricks the cell into believing that Gal is still present and thus causes it to continue silencing, so we can continue our experiments. Unless I misunderstood your question, your concern was that we would not be able to proceed with our experiments because the act of 'silencing' only lasts for 5 hours whereas the actual silencing of the target genes takes anywhere from 1~24hrs. Another thought occured to me while reading your blog, since we're plating on +Gal plates, wouldn't there actually be a constant stimulation because the plate.. contains Gal? which stimulates the Yeast Mating Pathway?

My Actual Blog:

I am most disappointed that my first project involving the testing of regional silencing is taking so long to complete. The original anticipated completion date was 7/3/08. But multiple problems arose, such as forgetting a simple step, which can determine the success or failure of your experiment, and the troublesome and time-consuming deduction that the Enzyme I was working with, Elongase, was 'broken'. But, now that we've figured out the problem, I am happily going on my way towards Success!

I've also realized recently that the Wiki contributes toward our scoring at the actual iGEM competition. I didn't know this last year, or at least, I might've heard about it, but didn't pay much attention to it. But now that it's 2A.M. in the morning, I've realized that updating this wiki isn't hard at all. It's quite fun when you have nothing else to do, or anyone to talk to =[

So, in conclusion for my first blog; I would like to simply remind people to try not to let reagents/items of importance sit out in the open when it wouldn't kill you to return it to the -20 freezer; enzymes and TOPO reagents especially. But almost everything you don't need should go back into your freezer. I've also been reminded a few times just this week. In fact, just a few hours ago... About 12 hours ago from the time of this blogging, haha. Thanks for the constant reminders Andrew. Sometimes it takes a few to get it through. It'll save me in the long run. Oh yeah, everyone should try to blog and update their wiki's. We Want To Win This Year's International Genetically Engineered Machines Competition.

~Jimmy

-[The Winner's Club] "Oh'Yeah!~"

0 comments

July 9, 2008 10:35 AM

by Andrew Horwitz

blllllllooooogggg....

Wow, I'm blogging. Jimmy, are you impressed?

I'm going to let the more experienced bloggers take over, but just wanted to pose a problem for you guys to think about--perhaps we can get a discussion going on her. Remember that silencing takes many hours to initiate (up to a day), and that when we do experiments with inducible LexA-Sir2, we plate the cells on +Gal media so that they have time to become silenced before we do the experiment. So here's the problem: signaling through the mating pathway is a transient event (Caleb tells me that the longest you can get the cells to signal through this pathway is 4 or 5 hours. This may not be long enough to set up silencing. We should probably do some experiments with transient Galactose stimulation (e.g. add Gal for a few hours, wash it out, then assay for silencing 24 hours later), but we should also consider ways that we might amplify or sustain the signaling through the mating pathway. In other words, how can we convert a transient mating pathway signal into a long enough signal to trigger silencing?

thoughts?

Andrew