Calgary/constructing wetlab

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Revision as of 05:32, 27 October 2007

The 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.

Work With The Light Sensor

The popularity of the light sensing biobrick parts since their release by the '05 Austin iGEM team is the best evidence of how useful a light induced system could be. Light can be easily and accurately applied with the right instruments, and could offer much more precise induction compared with the chemical inducers used today. However, just as the promise of the light sensor has spread, so has it's reputation as a tricky system to work with. Our project plan incorporated the light sensor controlling agarase expression, but most of our efforts on it were spent just trying to duplicate the functionality seen in the '05 Nature brief communication by Levskaya et al.

1) The Biobrick Light Sensor

On our first attempt to use the light sensor we used the Biobrick part M30109, which contains the three genes that code for the light sensor encoded in Biobrick format. M30109 was omitted from the iGEM'07 registry plate distribution by accident, so we made a special request for it from the registry.
The M30109-pSB1AC3 plasmid proved extremely difficult to work with, however. Routine restriction digests and PCR amplifications known to work with the pSB1AC3 plasmid backbone failed completely. CP919 is the light sensor chassis E.coli strain, and experiments with light to test the sensor in CP919 also failed. Ultimately, M30109 was set aside and a different source material for the light sensor was sought. However, before abandoning this part one last PCR probe was done using primers for each of the three light sensor coding regions (biobrick parts I15008, I15009 and I15010). The gel is included below, and it indicates that these three regions are intact in M30109. Why exactly M30109 fails remains a mystery, but given that our primers anneal at the restriction sites flanking Biobrick parts, and that restriction digests of M30109 didn't succeed, the current hypothesis is that a restriction site has mutated or is damaged. Given more time, we might have returned to M30109 to find out what's going on with it.

2) The Original Two Plasmid Light Sensor

After difficulties with the biobrick light sensor we moved on to the original version of the system: the one developped by Levskaya et al. Jeff Tabor, a former Austin iGEM team member, was kind enough to send us the plasmids pPL-PCB and pCPH8. Colony PCR of the pPL-PCB pCPH8 double transformant we made, using the proven light sensor coding region primers, revealed that the three genes were present, and that double transformation worked.

The next hurdle was to demonstrate that the system itself could work. CP919 transformed with both plasmids was incubated in light and dark, in the hope of seeing a light sensitive response but none was observed. On a tip from Jeff Tabor that the pPL-PCB plasmid may be unstable, we switched to using only freshly transformed cells in our experiments but this made no difference. Most recently, we acquired a red bandpass filer. A bandpass filter is a lens that allows only light of a certain wavelength to pass through it, our filter was selected to transmit only frequencies that the light sensor will respond to. Apparently, the sensor can be inactivated by infrared light, which the filter should block.

At the time of this writing our strategy is two fold: to experiment with more powerful light sources than the lamp we currently use, and to conduct more rigorous verification of the light sensor plasmids' integrity.

3) Biobrick Light Sensor Subparts (I15008, I15009, I15010, S03321, S03322, S03410, S03417, S03422)

While working with (and having trouble with) the full Biobrick light sensor M30109, we also entertained constructing a new copy of it from its subparts (listed). Many of these components proved to be just as unstable as M30109 itself, producing erratic PCR and digest results. Work on these subparts was dropped, but recently we returned to these parts as alternatives to one or the other of the original two plasmids. Construction had begun with biobrick parts S03410 and S03422, which contain between them the same genes as the pPL-PCB plasmid. Unfortunately this procedure was incomplete as of the end of the iGEM'07 work period, .