From 2007.igem.org

Projects

Design: Wet Lab

Design: Printer

Design: Software

Testing

Construction: The Wetlab

Protocols

Final Result of E.co Lisa

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 opposing part ompC. We decided to go ahead and begin testing OmpC, and to begin putting it together with other parts, just in case we had to use it. So every step noted above was done with ompC in place of ompF at the same time. And since ompC exists in two forms in the registry, (R0083 and R0082), both parts were used simultaneously. To test ompC, the part was put onto a GFP test construct, I13504 AC, to check the functioning and reliability of the promoter in both TOP10 and CP919 (the knockout strain needed for functionality of the light sensing system). After both of these new parts were made, they had to be transformed into both CP919 and TOP10. It was expected to glow in TOP10, though not too brightly, and to glow perhaps a little brighter in CP919.

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 needed to be tested, to see how tight its control really is. To do this, was attached to a GFP testing part I13401 AC, with a constitutive promoter placed in front. Since there should be no way to unlock the RBS in front of the GFP, the cells were not expected to glow at all, and this was indeed the case when we tested the construct out. At the same time the lock test was being constructed, the relevant key was attached to a constitutive promoter, and then this construct attached to the locked GFP. When transformed into E. coli, this construct was expected to glow quite strongly, alas the key proved difficult to work with, and this test has not yet been carried out. Each of these testing procedures was done in parallel, as there were two sets of lock/keys to test out.

Any crosstalk between the two sets of lock/keys also was to be tested. To do this, the key construct 1 would be attached to lock construct 3, and as well 3 onto lock 1. There should be no expression of GFP in either of these crosstalk experiments, or at least no more then the cells containing only a lock construct.

Finally, the rate of control needs to be characterized for the RNA lock/keys. To do this, an inducible promoter was to trigger expression of an RNA key, and for this we were going to use the AHL-induced promoter R0062. First though, we were in need of a standard control, made by attaching R0062 to the GFP testing construct I13504 AC. After that, a constitutive promoter was put in front of S01414 (which is necessary for functioning of the AHL promoter), and the two pieces put together. 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 was 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.