Project

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A Foolproof 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 module then the following illustrates the sequence of events that will take place:

Direct repeats
While if oriented in the same direction, 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 module.
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 module and appears as a circular module.
In other words, recombination at direct repeats causes a deletion.

Note that the reverse reaction -- the recombination of a circular module with another DNA module (either circular or linear), brings about a fusion of both modules or the integration of one module 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?
First, we define the Receptor as the vector that has already existed in the cell (E.coli.), and the Donor as the vector containing the desired gene that we intend to integrate into the Receptor. The gene circuits for these plasmids are illustrated below.

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 integrated into one circular DNA.

The recombinant DNA then could be selected in the liquid culture containing both ampicillin and kanamycin. 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 will preserve the killer gene ccdB, which is under the control of another inducible promoter. Because the two plasmids have shared origin site, plasmid incompatibility will occur thus the two kinds of plasmids will be separated into different cells.

When induced, CcdB could be expressed so that cells containing CcdB will be killed.

In order to realize the linkage of 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.

The reason for us to use two sets of attB/attP specific sites is to avoid the combination within one molecule.

 

The whole process

 

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. The manufacturing of Vitamin C in China, which has contributed to 60 percent of its world production, could serve as an excellent example to validate the significance of coculture in industry. 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.

The attempts to uncover the mechanics and complex interrelations within natural microbial systems and quantitatively measurement of environmental factors on system behavior often failed because of the entangled intrinsic parameters and un-measurable population dynamics.

We designed and constructed an ecosystem constituted of two strains of E.coli, which could represent various biological relationships along with the fluctuation of antibiotics concentration as the environmental pressure and the inducing molecular such as IPTG and arabinose as the regulating factors. 

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

The design for the cell I: The genetic circuit can be divided into three different functional sections.
The first one in the graph is the detecting Section. By using this section you can detect the cell density according to the intensity of the red fluorescence. The detecting section is especially useful when you incubate two different kinds of E.coli in a coculture.

The second section is the Helper section. We call it helper section because the LuxR protein is the prerequisite for the activation of PLux. Here we used a constitutive promoter to express the LuxR protein.

The core section is the toggle switch. Toggle switch is a genetic device that can switch between two convertible states, which, here, represents a different survival strategy for the cells each.

When adding Arobinose/AHL different genes will get expressed behind the two mutually-repressive promoters. That means when added into the culture AHL will diffuse into the cell bind the LuxR protein and form a complex which can activate the LuxPr promoter and then the genes of rhII capR and araC will get expressed. Then the araC protein will bind to the PBad/araC promoter and repress the expression of the aiiA and another capR gene. However, you can turn the switch to the other side by adding Arobinose. When adding arobinose into the culture, the repression functional molecular AraC protein will get released from the PBad/AraC promoter. With the expression of the aiiA gene the signal molecular will get digested and therefore decrease to a proper level which is not high enough to activate the LuxPr promoter.
The most important thing in this section is the capacity of the two different promoters LuxPr and PBad/araC are quite different. When the LuxRr promoter is activated, its higher capacity will express more chloromycetin resistant protein and another important thing is by sensing the AHL which is sent out by cell-two it can produce another kind of signal molecular BHL.
Cell 2 is similarly designed as Cell 1.

The design for the cell I: The genetic circuit can be divided into three different functional sections.
Cell 2 is similarly designed as Cell 1.

The first one in the graph is the detecting Section. By using this section you can detect the cell density according to the intensity of the red fluorescence. The detecting section is especially useful when you incubate two different kinds of E.coli in a coculture.

The second section is the Helper section. We call it helper section because the LuxR protein is the prerequisite for the activation of PLux. Here we used a constitutive promoter to express the LuxR protein. The core section is the toggle switch. Toggle switch is a genetic device that can switch between two convertible states, which, here, represents a different survival strategy for the cells each.

When adding Arobinose/AHL different genes will get expressed behind the two mutually-repressive promoters. That means when added into the culture AHL will diffuse into the cell bind the LuxR protein and form a complex which can activate the LuxPr promoter and then the genes of rhII capR and araC will get expressed. Then the araC protein will bind to the PBad/araC promoter and repress the expression of the aiiA and another capR gene. However, you can turn the switch to the other side by adding Arobinose. When adding arobinose into the culture, the repression functional molecular AraC protein will get released from the PBad/AraC promoter. With the expression of the aiiA gene the signal molecular will get digested and therefore decrease to a proper level which is not high enough to activate the LuxPr promoter.
The most important thing in this section is the capacity of the two different promoters LuxPr and PBad/araC are quite different. When the LuxRr promoter is activated, its higher capacity will express more chloromycetin resistant protein and another important thing is by sensing the AHL which is sent out by cell-two it can produce another kind of signal molecular BHL.

Mutualism and Competition

Competition

In the culture that both ampicillin and chloromycetin are available, it requires the expression of the both the resistant genes for both antibiotics for a strain’s survival. Without adding any signal molecular as the initially inducing factor into the culture the two kinds of E.coli can not communicate with each other so they will keep on the competent stage. In this stage each kind of cell must survive all by it self in some method as assimilating the nutrition in the culture. As a result the two different kinds of E.coli fight with each other for the space and nutrient ingredient.   

By adding some AHL into the culture, the LuxPr promoter will get activated by the AHL and LuxR complex. And then the expression product of the rhlI gene BHL will diffuse into the cell-two which can sense BHL-RhIR complex by binding to the PrhI promoter and turning on the expression of luxI kanR and lacI genes. The LacI protein will bind to the PBad/araC promoter and therefore stop the digestion of the signal molecular by the expression of the aiiA gene. At the same time the expression of the luxI gene will send out AHL .By using a very similar mechanism the cell-one can sense the AHL molecular.

Mutualism
In this state the two kinds of cells communicate with each other by sensing the signal molecular sent by the counterpart.
It seems that with the help of each other both of them can live better in the harsh environment and the fact is the capacity of the LuxPr and PrhI are higher the than the Plac and Pbad/araC promoters. With higher expression of the ampicillin and chloromycetin resistant protein both of them can survive in the ultra-high antibiotic concentration.

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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