Calgary/full procedure

From 2007.igem.org

Latest revision as of 17:26, 21 October 2007

EcoLisa is our Universities biological entry in the IGEM compeition. The goal of this project was to design a biomechanical printer. This printer works by using a laser to induce engineered E. coli produce agarase. The agarase will then dissolve the agar medium in which the E. coli is growing. The following procedure outlines the process our team went through to develop this project. A much simplified version of how our project works can be found in our Simplified Project Plan Section

Our team investigated a number of potential projects before selecting our light sensing printer. This section offers a brief outline of some of the ideas we had.

• Antibacterial Plasmid - A novel strategy for stopping difficult bacterial infections: give the patient an antibiotic and an engineered plasmid with resistance to that antibiotic. (Alternatively, give the bacteria any plasmid that would grant them a strong survival advantage in the patient.) Within a short time, most the infectious bacteria should be carrying the resistance plasmid... at which time a chemical signal is administered to the patient that initiates ccdB (control of cell death protein B) production on the plasmids, wiping out the infection.
• Hydrogen Disulfide Sensor System - A useful tool for the oilpatch might be a biological H2S sensor, in the form of bacteria that turn a different colour / glow in the presence of H2S gas. H2S is highly toxic; and the human nose cannot detect H2S at very low or very high (including potentially lethal) concentrations. This makes detectors invaluable for safety in H2S laboratories and in the oilpatch, where H2S is present in oil and especially in natural gas deposits. H2S sensors are not inexpensive however, a cheap biological sensor might be useful. Ironically, concerns about the safety of working with H2S in the lab precluded this idea =P.
• Addressable Bacterial Memory - This was a mostly unformed idea. The thought behind it was use previously developed bacterial memory to store something interesting or relevant to the bacteria.
• Phototaxic Bacteria - The idea behind this project was to couple a light sensing system to bacterial chemotaxis to create bacteria that woud swim towards the light
• Directed Evolution of the Light Sensor - The light sensor currently absorbs in a range around the 660 nm range: red light. Directed evolution could be used to modify this spectrum, possibly changing it to an entirely different light colour. Selective pressure would be applied by linking light sensor activation to antibiotic resistance gene expression, and gradually changing the emitted light frequency. As the frequency increases (or decreases), mutants with sensors sensitive to that frequency will produce more of the resistance gene product and tend to outcompete the rest in an antibiotic environment. Gradually changing the emitted frequency could allow us to 'pull' the actual absorbed frequency along.
• E.coli Reverse Chemotaxis - Another unformed idea. We were looking at setting up a system where E. coli would run away from nutrients
• Cross Species / Gram Negative to Gram Positive Signalling - One team last year (Rice U) attempted to do cross species signalling from a gram+ species of bacteria to a gram-, using a quorum sensed molecule. We beleive this experiment failed because gram+ bacteria have one membrane and gram- bacteria have two, and the particular gram+ signal was unable to penetrate to the engineered receiving system in the gram- strain. However, communication going the other way (gram- to gram+) shouldn't experience this complication.
• Use the Cell Specificity of Pathogenic Bacteria to Deliver Drugs - Certain pathogenic bacteria have evolved the ability to infect specific cell types in humans. If the toxin production in these bacteria was replaced with pharmaceutical production, the result would be a highly targeted drug delivery system. Probably some ethical issues with this one though...
• Isolate Bacterial Vesicles with Synthesized Drug Inside - The idea is to target a synthesized chemical / pharmaceutical to vesicles, small membrane sacs, within the cell. Then, the cells are lysed in a way that destroys the cell membrane while leaving the vesicles intact, allowing their easy recovery by centrifuge.
• Attenuated Cells: Iron Supply Limit - All cells require iron to live, and developping diverse iron acquisition systems is a particular passtime of bacteria. However, iron is used mainly in cofactors and enzymes: it is not consumed by the cell and is not excreted as a waste product. An engineered cell endowed with a large supply of iron but stripped of it's uptake mechanisms should be able to divide a finite number of times (each division halving the amount of cellular iron) before iron levels drop too low to sustain life. This might be good a mechanism for controlling genetically engineered species released into the wild, requiring them to die off after a set number of generations, thus limiting or negating their environmental impact. Ethical issues abound here too!

The project we selected was to design and build a biomechanical printer; composed of a two dimensional plotter equipped with a red laser, software to translate computer images into instructions for the plotter, and E. coli cells engineered to respond to the laser light. Bacteria are spread in a solid lawn on the plate, or mixed in the media before pouring the plate. The response triggered by this biological circuit will produce beta agarase, an enzyme which degrades the agar polymer that the cells rest on.

The printer can then be used to "draw" high resolution images on the bacteria with the laser. The bacteria will then dissolve the agar where the laser was shone. This results in Bacterial Lithography, where the dissolved agar forms a picture. Cool eh.

We also chose a second project to include in our entry to the competition this year. That is an in Silico Biobrick Evolution system. The purpose of this project is to design a system that will accept user entered parametres and use them to search through the registry database. Using the given parametres the system will try to construct circuts (a series of biobricks) that will produce the desired product.

The steps outlined in this section are the MOST CURRENT used in our project. This section does not describe the primers, plates, and parts that were considered but not used.

Preparing Reagents

Preparing Plates. AMP resistant

Have everything ready to go for when parts arrive

Quick list of parts...

• R0084 - Light Sensor Promoter
• R0062 - AHL Promoter
• R0011 - Temperature Sensitive Promotor
• S03600 - AHL Intermediate
• I13544 - GFP RBS Terminator
• J23008 - RNA Key
• B0034 - RBS
• B0015 - Terminator
• J06501 - Temperature Sensitive Component
• I51001 - ccdb and AMP resistant (death gene used to do plasmid switches

Wells from registery

I) Rehydrate the wells

II) 2 micro litres of rehydrated part --> transformed into TOP10

I) Colony PCR test...

• Design universal primers (valid for any biobrick out of any plasmid)
• Develop massive amounts
• Gel to test

II) Test Plasmids --> always grow up 5ml of overnight

• Plasmid Prep --> isolating plasmid out of the cell
• Sigma Plasmid Prep protocol

We are planning to build the simple system diagrammed above in order to eventually express agarase through laser activation of E. Coli. Since we have many of the parts in composite form, the goal will be to attach together the 5 composite parts above.

As of August 2 we have put together R0084 with A340620, as well as S01414 with I13504. On August 6 the plates of the parts need to be put into overnight liquid cultures, so that they can be plasmid-prepped on the 7th. Once we have the plasmids, we can put them together using the construction technique. After construction, this product will be transformed into both TOP10 and CP919 E. Coli using the transformation technique, and grown overnight. On the 8th we should have the finished simple system (except for the M part which is under construction still). It can be verified by adding AHL to the plates. After a few minutes, the cells (if they’re TOP10) should be glowing brightly green, while the cells in CP919 should not be.

When designing this system, there were some possible problems noted with a pivotal part in our system, OmpF (R0084), the promoter controlled by the light-sensing system.

There were some contradictions in the literature about what this part did, and exactly how it would respond to light. Also, this is not the part that the Texas team used in their project; they used the opposite part OmpC. We have decided to go ahead and begin testing OmpC, and to begin putting it together with other parts, just in case we need to use it. The part will first be put onto a test construct, I13504 AC, so that the functioning and reliability of the promoter can be tested in both TOP10 and CP919 (the knockout strain needed for functionality of the light sensing system). If the part proves viable, it will be attached to an inverter and then construction will begin using it.

Parts R0082 and R0083 need to be attached to I13504 AC, so they must first be taken from plates and grown overnight in a liquid culture on the 6th. On the 7th, the plasmids can be prepped out and each of the R parts may be attached to I3504 AC using the construction technique on August 7th. At the same time, the two promoters should be attached on to part A340620 AC, and these plasmids stored for possible future use.

After both of these new parts have been made, they need to be transformed into both CP919 and TOP10. It is expected to glow in TOP10, though not brightly, while in CP919 it may glow brighter. Both of these new constructs need to be saved until the M part is ready, and can be added to the end. Once this is complete, the three different Omp promoters can be tested against each other.

RNA Lock and Keys

One of the parts we were interested in using (for our off-switch) is an RNA lock and key to control translation. The lock first needs to be tested, to see how tight its control really is. To do this, it will be attached to a GFP testing part I13401 AC, and then a constitutive promoter placed in front. Since there should be no way to unlock the RBS in front of the GFP, the cells are not expected to glow at all. At the same time the lock test is being constructed, the relevant key needs to be attached to a constitutive promoter, and then this construct attached to the locked GFP. This new construct should be transformed into bacteria, and these cells are expected to glow quite strongly. Each of these testing procedures needs to be done in parallel, as there are two sets of lock/keys to test out.

Any crosstalk between the two sets of lock/keys also must be tested for. To do this, the key construct 1 needs to be attached to lock construct 2, and as well 2 onto lock 1. There should be no expression of GFP in either of these crosstalk experiments, or at least none more then the cells containing only one lock construct.

Finally, the rate of control needs to be characterized for the RNA lock/keys. To do this, an inducible promoter needs to trigger expression of an RNA key, and for this we will be using R0062. To do this, we first need a standard control. R0062 will be attached to I13504 AC. After that, a constitutive promoter needs to be put in front of S01414 (which is necessary for functioning of the AHL promoter). This construct should not glow in TOP10, until AHL is added, and the speed of this expression should be characterized.

During construction of the lock pieces, another part should be made by putting S01414 (RBS and LuxR) behind the locked GFP, and before the terminators. This construct should not glow on its own, but should after addition of AHL.

Construction Schedule

Monday August 6th - Over night cultures made for several parts

• R0084+A340620 AC
• S01414+I13504 AC
• J01080+I13401 AC
• R0082
• R0083
• J23008
• R0040

Tuesday August 7th - plasmid preps of all of the over nights started on Monday August 6th
Attachment of...

• R0084+A340620 (as insert) and S01414+I13504 AC (as vector)
• R0082 (insert) and I13504 AC (vector) [Into TOP10 and CP919]
• R0082 (insert) and A340620 AC (vector)
• R0083 (insert) and I13504 AC (vector) [Into TOP10 and CP919]
• R0083 (insert) and A340620 AC (vector)
• R0040 (insert) and J01080+I13401 AC (vector)
• J23008 (insert) and B0015 (vector)
• S01414 (insert) and B0015 (vector)

• J01010
• J01008
• E0040

Wednesday August 8th - Overnights of the transformed and constructed parts from yesterday.
Digest with Not1 and then run on a gel to verify the lengths of the plasmid preps done yesterday

Thursday August 9th - Plasmid preps of all of the overnights.
Attachment of...

• R0040 (insert) and J01010+I13401 AC
• R0040 (insert) and J23008+B0015 (vector)
• J01008 (insert) and B0015 (vector)
• J01010 (insert) and I13401 AC (vector)

Friday August 10th - No1 digest and gel verification of all of the plasmid preps done on the 9th

prepare bacterial broth then pour as agar.

Set up printer system