From 2006.igem.org

Introduction

Our brainstorming meeting on 3-16 gave us several preliminary ideas which we will explore via modeling and research

Things I learned at the teach the teachers meeting

Standard plasmids: pSB1AK3 ("ampicillin kanamycin")

                 pSB1AT3 ("amplicillin tetracycline)
                 pSB1AC3 ("ampicillin chloramphenicol)

all of these are high copy number, if inappropriate, also provided are inducible plasmids:

                 pSB2K3 (IPTG inducible)

the "3" refers to the MCS and the transcriptional terminator We will be provided with a plate with dried DNA samples, we can amplify the ones that we want There are standard cloning protocols that use specific sites; products are checked by sequencing with promoter sequences VF ("verification forward") and VR ("verification reverse")

They often use 3-way ligations selected with 2 antibiotics to create composites You can put > 1 plasmid in a cell; chose according to copy number desired (eg, you might want GFP at high copy and AraC at low...)

EVERYONE should have a BioBrick account with their real name go here: http://partsregistry.org/Help:Create_a_Registry_Account It is important to document parts every time we create them They should have the "BioBrick ends" what does this mean?

We will get 2 384-well plates with the DNA; pierce the foil, dissolve in 30-50 microliters, and transform it's colour-coded with food coloring: KANAMYCIN = red; TET = yellow; CHLOR = green; AMP = orange the key to what's in each well will be provided on the registry fill out address on Group pages next week! when we send parts, they should be sent as stabs labeled w/ part name, plasmid, cell (on Help page) (or foreign schools can send dried DNA) as soon as a part is Available, it will be sent to EVERYONE, that way they don't worry about shopping lists The letters on the plates are very small, so watch out

Discussion

Hi eveyone! Since we're not going to have a meeting for a while, I thought it would be a good idea to post our ideas here. - Horia

Alright, it's 1:39am...and I've come up with some random idea as I was studying for my bio exam. During the last meeting, we talked about pH and voltages to control protein regulation. Would it be possible to connect those two ideas (pH and voltages)? I am pretty sure the intracellular function changes as pH changes (like in plants, certain enzymes get activated as pH drops after hydrolysis done by certain hormones) and this might have some effects on the voltage gated channels on E.coli and allowing different influx/eflux of ions that would manipulate activation of protein synthesis. I haven't looked up on the web to see what exactly E.coli do in different situations yet..but, I wasn't really too keen about using wire to generate voltage on that little E.coli...the voltage idea itself is brilliant, and I thought this can be incorporated into pH with the use of voltage gated channel...OR, pH(input) can regulate the voltage difference (output)to create different musical notes (referring back to the last meeting)..Alright, back to studying. - Jieun

Hum, 1:54 am, so it would either be " Note -> Voltage -> pH -> Function" or " Function -> pH -> Voltage -> Note". I guess the question is how to transform voltage into pH or vice versa. Also which ion channels would open in response to a change in pH and how that would affect function. I remember Belinda (correct me if i'm wrong) saying something about using a vesicle filled with a chemotactic agent whose pores would open in response to applied voltage. Maybe if we filled them with carbonic acid or something that would do the trick. I read something similar here (with glucose): http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6261800&dopt=Abstract Josh talked about using a keyboard and i thought that if we somehow drew a small musical staff on the bottom of the petri dish, we could have the bacteria cluster at different points on the staff depending on which notes we played. Thus we'd write out the music! We could have an electric grid or just individual electrodes that would determine every possible point, and these would trigger the release from (anchored) vesicles of an aggregating agent ( Sodium Fumarate?: http://web.mit.edu/biophysics/papers/PNAS2003b.pdf). To have the cells cluster at the right spots we could have small wells for each horizontal bar on the staff, so that the next note won't influence the previous cluster (i'll try to draw a picture tomorrow). I don't know how big the clusters get so I don't know if this whole thing is feasable... We also talked about using an oscillator (between two colours?) to do a sort of light show. I guess I'll have to read more about those.. It wasn't in any bio class that I took, that's for sure :P - Horia

Hi- I was just searching the internet for genes in e-coli that are senstitive to pH changes and I happened to read about the yfid gene. The article basically said that expression of this gene (which was attached to a reporter gene) was upregulated 3-5 fold when placed in an acidic environment during anaerobic conditions. For example, when the pH of the cytoplasm reached 4.4 the expression of the gene was strongly induced. So therefore, I was thinking that perhaps attaching a GFP gene or some other flourescent gene to the yfid gene could help us visualize pH changes. The link to the article is http://mic.sgmjournals.org/cgi/content/full/148/4/1015 -Ashwini

That's pretty cool. I guess it would help in the pH->function part if we're going to do anything along those lines. Also, I was wandering if anyone was going to go to the meeting tomorrow. If so, could someone post what was said for those of us that can't make it? That would be really appreciated, thanks! -Horia

Meeting on April 20th-

1. We first just went over what "bricks" are. So they are bascially promoters, genes, regulatory seqeunces that are going to be ordered from MIT.

2. We then started talking about how bacteria can be made sensitive to voltage changes in accordance with the idea presented last week concerning the conversion of sound to voltage. One of the proposed ideas was the use of voltage gated channels in response to a certain threshold voltage. The advantage of this is that the voltage gated channels open quickly and can potentially result in a continuum of current. However, how can an influz of cations translate into something visible? The possibility of proteins sensitive to ions was discussed however there aren't many of these proteins. In addition, the possibility that a cation influx could result in the tranlation of proteins was also introduced yet the problem with this idea is that transcription/translation of proteins is too slow a process for our needs.

3. Two voltage gated channels that Prof. Nadeau has in the lab are MSCL which is sensitive to pressure changes, and NACHBac which is sensitive to voltage changes.

4. The idea of using bacteria to produce a picture was reintroduced. One of the past projects had taken advantage of light sensitive bacteria to produce a picture, and therefore, for us to explore this idea the bacteria has to be sensitive to something other than light when producing the picture. Some sugars like lactose and arabinose as chemical triggers in the production of color in bacteria was suggested. However, diffusion of the chemical triggers and the secreted color would be too fast to obtain a clear image.

5. The idea of putting a catylase into e-coli resulting in the production of oxygen and water was also discussed.

6. There will not be a meeting next Thursday, April 27th. The first day of the Biomedical Engineering 506 class will be on May 2nd and we will meet in room 321 in the Duff building. The class starts at 10 and ends at 1 on MTWR. Prof. Nadeau is trying to get the fees for the summer course waived for iGEM participants by negotiating with certain science departments. So please email Prof Nadeau the department that you are part of as well as the contact information for the head of your department in order to waive the summer fee.

7. The articles "Reconstruction of Genetic Circuits" by David Sprinzak and Michael B. Elowitz, from Nature (November 24 2005,Vol 438) as well as the article "Combinatorial Synthesis of Genetic Networks" by Calin C Guet, Michael B Elowitz, Weihong Hsing and Stanislas Leibler from Science (May 24 2002, Vol 296) were handed out to read.

-Ashwini

Here is a silly idea for creating a picture with bacteria:

Crayola Bugs

We have several adhesion tags. If we "fusion clone" them to something that is on the outside of the cell (use part of NaChBac to anchor, maybe?) the cells will be able to stick specifically to certain targets. We could stick beads coated with glutathione, biotin, cellulose, and carboxy-resin in a dish to draw a picture. Then add bacteria like GST-RFP, Streptavidin-YFP, CBP-GFP, Arg-CBP. They should preferentially stick on surfaces coated by their targets, and colour in the picture. If we wanted to be really cool about it, we could have induce the colour with light, heat, cold, or lactose - or even set them off one after the other...or make them set off each other: the red one secretes something to set off the yellow one, which then secretes something else to set off the green one, which in turn activates the cyan bugs. Actually, yellow, green and cyan are pretty close so we may want to just use red, yellow and cyan, which should be distinguishable.

Dyslexic Dinosour, May 3, 2006

P.S. I just finished sorting out which bricks could be potentially useful, they are on the shopping list on the main page (I sure hope they are free...)

Ok, I just had an idea about combining the picture feature with the oscillator feature. What if we could oscillate between two pictures, or words (like those panels that have alternating advertisements)? The oscillator would oscillate between two substances that would each activate one of two fluorescent proteins. First, we create two pictures with two different anchoring beads on the same plate. We add E.Coli with adhesion tag 1 and RFP that stick to bead-picture 1, E.Coli with adhesion tag 2 and YFP that stick to bead-picture 2 and E.Coli with the oscillator. The oscillating bacteria release Substance 1, which activates RFP in picture 1, then Substance 2,which activates YFP in picture 2, this way we get sequential activation (and degradation) of the red and yellow, thus alternating between the pictures. The oscillators should be all synchronized, and we have to have substances that activate yfp & rfp specifically and which can be secreted by e.coli. Maybe we could use two different Luciferin/Luciferase pairs because I think this would give a faster response (but how fast can the oscillator be?) In any case, tell me what you think. Oh, and could someone please post the name etc. of the new article she gave in class the other day? -thanks

-Horia

IMPORTANT INFO ON MSCL AND THIOREDOXIN (May 16th, 2006) follow the link and download the full text http://www.jbc.org/cgi/content/abstract/273/41/26670 (J Biol Chem, Vol. 273, Issue 41, 26670-26674, October 9, 1998 Release of Thioredoxin via the Mechanosensitive Channel MscL during Osmotic Downshock of Escherichia coli Cells)

http://www.jbc.org/cgi/content/full/271/36/21956 (Volume 271, Number 36, Issue of September 6, 1996 pp. 21956-21968 ©1996 by The American Society for Biochemistry and Molecular Biology, Inc. The 1.5-Å Resolution Crystal Structure of Bacterial Luciferase in Low Salt Conditions*)

http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WBK-45FK7XF-5K-1&_cdi=6713&_user=458507&_orig=search&_coverDate=12%2F09%2F2000&_qd=1&_sk=997209998&view=c&wchp=dGLbVtz-zSkWA&md5=98385089cd9ca167699460c8ad2bdf25&ie=/sdarticle.pdf (Biochemical and Biophysical Research Communications Volume 279, Issue 1 , 9 December 2000, Pages 208-212 Use of Small Fluorescent Molecules to Monitor Channel Activity) -Jieun

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Hey guys,

I hope everyone's having a great holiday weekend. Well, I've been trying to see a big picture as to what we are trying to establish in our project, and I've come across some questions. I think it'd be great if I can get some feedbacks or, even answers to these concerns.

>compatibility of luciferase and EcoMScL (would luciferase be small/large enough to go through EcoMScL?)

>a heterodimer type of luciferase has alpha and beta subunits (40kDa and 37kDa respectively), and a literature said alpha subunit has the active site, does this mean it would be possible to reduce the physical size of luciferase by only having alpha subunit? what does beta subunit do anyway?

>activation/reaction pathway of luciferase and luciferin

>binding time (if any) of luciferase and luciferin (this may be critical for the "oscillation" we are trying to achieve)

>degradation of luciferase-luciferin complex (again, for the oscillation)

>promoters that would also incorporate Lux genes somehow (as far as I know from the articles I've read,Lux genes encode proteins that are necessary for the luciferase reaction pathway)

Well, that's all for now...and have an awesome holiday weekend! - Jieun at 1:05am

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