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Summary

Our project is based on the fundamental concept of plasmid instability in a novel way to conceal information or ‘lock’ a gene’s function in a cell until the correct combination of inputs is fed into the cell. We call this a ‘combinatorial lock’ or PLASMID. It involves the positive regulation of the gene of interest only on receiving the correct inputs from the user. We use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way (i.e, essentially designing a genetic circuit) so that they repress the expression of the gene of our interest. As the selection pressure is lifted from the media, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes. In essence, the process of unlocking would simply be the correct sequence of antibiotic media in which the cells should be washed. We would be working with a 2 plasmid system and it is easy to see that this principle, theoretically, could be extended to N plasmids. In general the code length required to "unlock" is N-1 if the number of plasmids introduced are N. In our case, since the number of plasmids being introduced are 2, the code would essentially be just 1 unit long. Particularly in this case, the 1 unit of code corresponds to growing the cells in one correct antibiotic medium.

Fig1: picture..picture...picture...picture..picture (of a network of plasmids and a gene of interest being represed)

However, in the experiments we have not incorporated any particular gene to be repressed. instead we study how can we achieve a directed loss of plasmids which is the idea central to the working of the system. In place of a gene of interest, we have placed fluorescent reporters in each plasmid to monitor the presence or absence of any particular palsmid.

Theory

The most important idea behind the working of the lock is plasmid loss due to lack of selection. Any extra-chromosomal genetic material introduced into the cell tends to disappear over the generations, unless they confer a selective survival advantage over the cells that do not possess the plasmid. During the growth of bacteria, plasmid-free variants arise in the initially homogeneous plasmid-bearing cell population basically in two ways. First, each plasmid-bearing cell has a certain probability to give rise to a plasmid-free cell at cell division (this depends on the mechanisms of plasmid distribution between daughter cells, plasmid copy number at the cell division, the presence of multimer resolution loci, etc.).

Fig2: picture..picture...picture...picture..picture (of cells dividing and one daugher cell not having hte plasmid)

Fig 2.1: picture..picture...picture...picture..picture (if possible put some kind of differnce in growth curves of cells with plasmdi and cells without plasmid)

Usually, such probability is very low for natural plasmids (about 10-7) whereas recombinant plasmids (i.e. genetically modified) may segregate with a higher probability (10-3—10-5). Several hypotheses have been put forward to explain this tremendous difference: impaired copy number control, the absence/impairment of the multimer resolution genes and random distribution of plasmid among daughter cells for low copy number plasmids. Second, it was experimentally found that plasmid-bearing cells usually have a lower maximum specific growth rate than their plasmid-free counterparts, and once a plasmid-free cell arises, it competes with its plasmid-bearing counterparts very rapidly and ultimately phases it out. Since most recombinant plasmids are not conjugative (not capable of self-transfer to other plasmid-free cells), if a cell has lost a plasmid, there is no way for the cell to acquire it again. Thus, a segregation of plasmids at cell division and the difference in the growth rates of plasmid-free and plasmid bearing subpopulations determine the rate at which plasmids are lost during prolonged cultivation. Here, we use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way so that they repress the expression of the gene of our interest. As the selection pressure is removed, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes.

Fig3: picture..picture...picture...picture..picture (a series of pictures with decreasing number of plasmids and the last pic showing only the required plamsid and expressing hte gene of interest)

Circuit

One of the plasmids will have the gene of interest which will be expressed only when the plasmids apart from this particular one are lost in a particular order. This particular order or “code” will consist of a sequence of antibiotic treatments given to the transformed cells. The correct code triggers the loss of plasmids in the cells in a particular order. Each plasmid will be linked to the plasmids which are supposed to be lost before and after it in a very tightly regulated fashion. The regulation of the plasmid loss can be regulated very tightly using a “suicide gene” (gene coding for the bacterial gyrase poison). These genes will be triggered when the culture is subjected to the wrong antibiotic (Out of sequence). To ensure that the suicide genes don’t fire randomly, they are under the control of repressors which are on the plasmids that are supposed to be lost after it and are expressed constitutively. Thus, they fire only when the plasmid containing the repressor is lost. When all the plasmids other than the plasmid containing the gene of interest are lost, the repressors that have been blocking the required gene are lost, thus allowing its expression, which can manifest as a phenotype or a function in the cell. This will be the “unlocking” of the lock.

The grand idea of a 3 plasmid locking system revolved around constructing something like the below

fig3.1: FIG fig fig

To which we thought we could fit in the following parts

Then we scaled it down and began to build constructs for a 2 plasmid system which works like this

To which we fit in parts from the registry to make it look like this

For the proof of concept experiments, we built the following constructs

[http://partsregistry.org/Part:BBa_K272001 K272001-constitutive RFP expressor]

[http://partsregistry.org/Part:BBa_K272002 K272002-constitutive CFP expressor]

Expected behavior of the system

Fig4: picture..picture...picture...picture..picture (2 plasmid case)

Fig5: picture..picture...picture...picture..picture

Potential Applications

One can think of a number of potential applications of this system of ‘combinatorial locks’, an example being the field of medicine. Human body releases a myriad of chemicals everyday in a certain order according to external stimuli or otherwise. In case of a disease, if this order goes awry, then this lock can be used to release the required chemicals to bring back the desired equilibrium. Also, when one wants to have the expression of a gene of commercial interest available only to licensed users and not a third party, this lock could be used. This has profound implications in the field of medicine in terms of regulated gene therapy and tissue specific control of drug release. The locking plasmid circuitry can be engineered to be incorporated into higher cells like our own. Such a system will detect the patterns of substances that are secreted by the cells and can activate a response if any link in a pathway has gone out of the way. This mechanism can also be used in bioreactors in industry, where certain functions of the bacteria are needed at specific stages of the fermentation. For instance, if a certain compound needs to be produced only after the culture reaches a predetermined population density, then the locking mechanism can be linked up a cell density related parameter for the timely secretion of the protein or a molecule of interest.

Work Plan

Building the constructs

We have been following 3 different strategies to build our constructs. 1) The 3A assembly

2) The 2A or Standard Assembly 3) PCR based approach: We plan to proceed with the experiment using a PCR based amplification system, followed by a specific restriction digest and subsequent ligation.

working with the construct

We will be performing a proof-of-concept experiment using the 2 plasmids - K272001 and K272002. The cells will be co-transformed with the plasmids. Each of these plasmids have a different fluorescent reporter and their backbones have different antibiotic resistances. However, the origins of replication in both the plasmids are the same, thus creating a tight competition between the plasmids to remain in the cell based on the selection pressures. The bacteria "titrates" the number of plasmids with a certain origin of replication before each replication. Thus if there are a couple of plasmids with the same origin of replication, and one of them happens to be non essential and does not confer any advantage to the cell for a given growth condition it will eventually be phased out from the population. This is essentially mimicking natural selection. The fluorescent reporters will help in tracing which plasmid is phasing out for a given set of growth conditions.
The cotransformed cells will be grown in different Antibiotic media and the loss of each Plasmid will be studied using fluorescent markers under a microscope. Presently we are working with the K272001 (constitutive RFP) in pSB1C3 and K272002 (constitutive CFP) in pSB1A2. We expect to see that the cotransformed cells when grown in a medium with both chloramphenicol and ampicillin should have more number of cells with both the fluorescent reporters than with those which are grown in medium containing either or none of the antibiotics. Specifically, if the cells are grown in medium containing only ampicillin, then the number of cells without CFP should increase and when grown in the absence of chloramphenicol, the number of cells without RFP should be on the rise.

pic..pic...pic..pic..pic...

Experiments

3A assembly

Standard Assembly

PCR based Approach

Results