Calgary/constructing wetlab

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Revision as of 04:30, 26 October 2007

Logic Circut

We planned to build the simple system diagrammed above in order to eventually express agarase through laser activation of E. coli. Since we had many of the parts already in composite form, the goal was to attach together the 5 composite parts above.

Construction Outline

When constructing a composite part, the two pieces can be classified as an insert and a vector. One piece is entirely cut out of its plasmid (the insert), while in the other plasmid (the vector) an opening is made just in from of the coding region itself (this is backwards in a reverse construction, which we did not use). After the ligation step in the construction, there are several different plasmids in the mix. First, you may have original parent plasmid that was never cut, from both plasmids. The probability of having parent vector plasmid is quite small though, due to the phosphatase treatment in the construction. You may also have insert plasmid that has had the actual insert cut out. Both this and parent insert plasmid will confer a certain antibiotic resistance to any bacteria that will uptake it, and as a result, you will have no way of knowing which bacteria have uncut insert plasmid, cut-out insert plasmid, or your desired construction product. This problem is why it is important that the two parts you are joining together have different resistance markers in their plasmids, or at least that the vector plasmid has a resistance the insert does not. Looking at the four composite parts above that we were planning to use, all of them have only ampicillin resistance. Because of this, before we could start any constructions, we had to confer a new resistance to two of the above composites, and this required a plasmid switch. These involve two items: a plasmid with the required resistance and containing the cell death gene ccdB, and the part you wish to switch. All you need do then is mix the two parent plasmids together, insert the appropriate enzymes to cut out both ccdB your gene, and then ligate. There will be four possible products from this procedure. If ccdB ends up in either its original plasmid or in your old plasmid, any cell that uptakes it will die. If your part ends up in its old plasmid, it will be killed, as it will not have the resistance genes of the new plasmid. Therefore, the only cells that survive will be the ones containing your part in the new plasmid. Plasmid switches were done with parts and I13504 and A340620, moving them into plasmids containing ampicillin and chloramphenicol resistances, as these were to be the vectors in the subsequent construction techniques.

With our two newly modified parts ready, the first step was to attach together R0084 with A340620, and S01414 with I13504. Once the construction of these two composites was complete, overnight cultures were made, and the plasmids isolated. The plan was to then attach together these two composites, but again they both had the same resistance markers. To overcome this problem, we again did a plasmid switch, moving the new composite S01414 + I13504 into a plasmid containing ampicillin and kanamycin. We were then able to attach together our two composites into our final logic circuit.

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