Project

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A Synthetic Plasmid Self-Assembly system
Background

Site-specific recombination
Site-specific recombination differs from general recombination in that short specific sequences which are required for the recombination, are the only sites at which recombination occurs. These reactions invariably require specialized proteins to recognize these sites and to catalyze the recombination reaction at these sites.

Inverted repeats
If the two sites at which recombination will take place are oriented oppositely to one another in the same DNA molecule then the following illustrates the sequence of events that will take place:

Direct repeats
If the two sites at which recombination will take place are oriented in the same direction in the same DNA molecule then the following illustrates the sequence of events:

The net result is that the segment of DNA between the two recombinogenic sites has inverted with respect to the rest of the DNA molecule.
In other words, recombination at inverted repeats causes an inversion

The net result is that the segment of DNA between the two recombinogenic sites has been deleted from the rest of the DNA molecule and appears as a circular molecule.
In other words, recombination at direct repeats causes a deletion.

Note that the reverse reaction -- the recombination of a circular molecule with another DNA molecule (either circular or linear), brings about a fusion of both molecules or the integration of one molecule into the other. The integrated segment will be flanked by directly repeating sequences which can, of course, be used to excise the integrated segment again.
Integration of bacteriophage lambda

In order for the lambda prophage to exist in a host E. coli cell, it must integrate into the host chromosome which it does by means of a site-specific recombination reaction.

The E. coli chromosome contains one attachment site which is designated attB. The site is only 30 bp in size and contains a conserved central 15 bp region where the recombination reaction will take place. The structure of the recombination site is usually represented as BOB'.

The bacteriophage recombination site - attP - contains the identical central 15 bp region as attB. The overall structure can be represented as POP'.

Integration of bacteriophage lambda requires one phage-encoded protein - Int, which is the integrase - and one bacterial protein - IHF, which is Integration Host Factor. Both of these proteins bind to sites on the P and P' arms of attP to form a complex in which the central conserved 15 bp elements of attP and attB are properly aligned.

The result of recombination is that the integrated prophage is flanked by two attachment sites but now they are slightly different: attL has the structure BOP' and attR has the structure POB'.

Cre-Lox recombination is a special type of site-specific recombination, which is often applied as a gene knockout tool.
Cre is a site-specific DNA recombinase, which can catalyse the recombination of DNA between specific sites, e.g. loxP in a DNA molecule. When cells that have loxP sites in their genome express Cre, a reciprocal recombination event will occur between the loxP sites. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase. The efficiency of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion, while a direct repeat of loxP sites will cause a deletion event.

Lox P site
Lox P (locus of X-over P1) is a site on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.
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Objectives: Bacterial assembly is aimed to be achieved based on the mechanism of site-specific recombination systems, So that the expensive reagent as well as the laboring tasks could be saved in gene cloning experiments.

Our design

We have innovatively utilized the site-specific systems mentioned above to build a foolproof bacterial assembly system to future reduce the labor and cost involved in gene cloning experiments. We have designed three standardized vectors which perform as the donors, receptor vector respectively.

How do they work?
When the donor vector carrying the gene of interest GENE1 was introduced to the E Coli which contains the Receptor vector, the site-specific recombination will occur between the attB1 site and the attP1 site, so that the two sequences will be integraded into one circular DNA, and then, under inducible conditions, Cre will be expressed and the recombined sequence will be divided into two separate plasmids; one will retain the desired gene 1, while the other preserves the killer gene ccdB, which is under the control of another inducible promoter. When induced, the promoter will express CcdB so that cells containing CcdB will be killed. In order to link GENE 1 with GENE 2, we will introduce the new plasmid containing the desired GENE2 to the survival cells, in which the plasmids containing GENE 1 will behave as the new Receptor plasmid. Very similarly recombination between the attB2 and attP2 and the cleavage between the two loxp  sites will be performed, and plasmids containing the linked GENE1 and GENE2 will be selected when the promoter expresses CcdB is induced.

Where have we been?

The synthetic convertible ecosystem

Background
There is no mono-culture in nature! And in industry, coculture of species/strains are widely used to either improve productivity or lower the cost, thus to understand the interactions between coexistent ecosystems will not only contribute to human’s perception of nature but also to human practices in engineering.

Most of the existent ecosystem could not be simply defined as symbiosis or competition, for instance. A huge amount species living in a symbiosis ecosystem will somehow compete with each other for food and space or other, such as the bacteria living in human intestine. The interweaving and intricate relationships in natural coexistent ecosystems shadow the human endeavor to deeply understand the dynamics of symbiosis or competition ecosystems. Thus a lot of effort has been made to fabricate a simplified ecosystem to emulate natural ecosystems. Approaches like auxotroph have been applied to achieve this goal.  However, most of the natural coexistent systems are based on cell-to-cell communication mechanisms, among which, quorum sensing plays a large role, which is one of basis for our built.

We aim to build an ecosystem, the relationship within which could be regulated by culture conditions.

The Tools

Toggle switch-----toggle switch is a switch on the basis of two mutually-repressive promoters, the product of each represses the express of that of the other, and both the repressors could be deactivated in certain conditions. And the state of the cell could be regulated by the change of the culture variations.

Quoru m sensing-----Th at is the way how various bacteria “talk” to each other. It is the mechanism ensures that certain genes will keep silent before the cell density of the species pass a threshold.

Prisoners’ Dilemma----It is the dilemma in which the two suspects could either choose to cooperate with or betray each other. In conditions when they could communicate freely with each other, they will cooperate, which maximizes their benefits as a whole; while when they are inquisited separately, they will both choose to betray one another to lower the risks of long sentence.

  Our Design

We aim to build an ecosystem, the relationship within which could be regulated by culture conditions. To realize this goal, toggle switches have been built to regulate the interactions between the two strains. As is shown in the illustration above, we used kanamycin and chloramphenicol as the selective forces.

When auto-inducers, either AHL or BHL is introduced to the culture, LuxPr or Prhl will be activated to produce the auto-inducers required by their partners, and express the anti-biotic resistant genes to ensure each other’s survival. During the process, PBad/arac and Plac will be repressed because of the repressors Arac and LacI expressed by LuxPr and PrhI.

However, upon the introduction of IPTG or arobinose, the repressors will stop functioning, so that Aiia will be expressed, and therefore AHL and BHL will be degraded, so that there will be no communications any more, and the relationship between the two strains will enter the phase of competition.

This idea was inspired by the theory of Prisoner’s Dilemma. As in prisoners’ dilemma, the bacteria in our design are faced with two solutions for coexistence, they could either choose to cooperate with one another by providing inducers to express their partners’ antibiotics-resistance genes or they could take a foe strategy in which no cooperation is needed for both strains’ survival.

  
       

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