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Team:HKUST-Hong Kong/asm.html
From 2011.igem.org
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STRAIN CONSTRUCTION
1. Constructing EX – the bacterial strain that allows selection without use of antibiotics
To study the population dynamics and behavior of a certain antibiotics sensitive strain of E Coli in a medium of antibiotic, our E. Trojan that is introduced into the culture medium must not process a wide spectrum of antibiotic resistance that impose a selective advantage. At the same time, E. Trojan needs to be transformed with the T4MO gene to carry out its job of signal disruption.
Summarizing the above criteria, a solution where the bacteria can be transform with the gene of interest while remaining sensitive to antibiotics is needed. Therefore the requisite is to construct a new bacterial strain that can perform plasmid selection without the use of antibiotics, and contains as little antibiotics resistance gene as possible.
With advancements in molecular cloning, nowadays different selection methods are available for different purposes. Application of antibiotics as the external selection pressure remains the most common and routine method, but it might interfere with the results in our study of relationship between MICs and indole degradation. Selection systems that generate internal stresses, such as those that base on auxotrophy and toxin-antitoxins interactions, are also available. Yet, nutrient statues and toxicity suppression might introduce more variables. Thus, we propose the idea of constructing a new strain of bacteria EX, that perform selection from internal pressure on holding an essential gene. Our EX might eliminate use of antibiotics in cloning, simplifies transformation and hopefully proof itself useful to the versatile field of selection methods.
2. How to select against EX without the vector plasmid? Our alternative selection method
Our EX will have one of its essential genes (genes that are required for viability) removed from its genome, and relocated onto an engineered plasmid pDummy. As illustrated, in order to survive, EX must rely on those extra-chromosomal copies of the essential gene; therefore, EX is addicted to pDummy. By having direct control over the replication of pDummy, we dictate the life and death of EX (and hence the name pDummy).
Here, we introduce a heat-sensitive origin of replication as the only origin of pDummy. When we intend to switch off the replication of pDummy, we can incubate EX at above 30 degree Celsius. This origin would then cease to function, and pDummy cannot be maintained. Deprived of the essential gene and the corresponding vital product, EX cannot propagate, unless, it receives an alternative but heat insensitive analog of pDummy.
This analog, named pCarrier, is the essentially our vector in cloning. Under an unfavorably high temperature, only those EX that are transformed with the insert-bearing pCarrier will be able to propagate and survive, while the others cannot undergo division and are virtually eliminated from the population. Eventually, the pDummy can be considered to be "shuffled out" by pCarrier. Our designed selection system, in short, bases itself on plasmid shuffling, and thus eliminates involvement of antibiotic resistance genes in any of the cloning steps.
3. Stepping in the heart of construction - methods of assembly
3.1 Construction and maintenance of an antibiotic-resistance-gene-free plasmid through antibiotic selection – the unavoidable evil two plasmid system
Our ultimate goal is to construct our EX without conferring it any new antibiotic resistance. For this reason no resistance gene should be found in our dummy plasmid pDummy.
Yet, such a plasmid would not be maintained by itself unless the host bacterium develops an addiction to it (i.e. losses the essential gene in its genome and depends on extra-chromosomal copies on pDummy), and inconveniently, the addiction can only be achieved after the introduction of the plasmid.
The solution is to develop a mutualistic relation between two plasmids and we planned to exploit positively regulated origin of replications.
Well studied examples are those in pSC101 and R6K origins of replication, where the origins of replication (OR) appear together with a constitutive gene (G). Initiation of replication happens if and only if the trans element of the gene is provided.
Let’s consider the following scenario:
i. G is placed on pDummy with no selection marker but with a normal replication origin
ii. OR is the sole origin of replication of another plasmid (here we introduce a new plasmid pToolkit) with a selection marker
iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress
We would obtain three possible outcomes:
1. only pDummy is uptaken
- since pDummy has no selection marker, the host bacteria die under selection pressure and cannot propagate
2. only pToolkit is uptaken
- the host bacterium that uptakes pToolkit survives. Yet during propagation, pToolkit is not replicated because proteins of G are absent. Therefore daughter cells of the host bacterium will not receive copies of the pToolkit and die under selection pressure.
3. both pDummy and pToolkit are uptaken
- in presence of pDummy, pToolkit is maintained and confers the host bacterium with stress resistance. Daughters that receive copies of both plasmids will survive and eventually develop into a colony.
Using this mutualistic relation, the desired pDummy can be maintained once the host bacterium develops an addiction it, and pToolkit can be lost in bacteria propagation if the expression of G can be shut off manually. Eventually, the bacteria not obtain any new antibiotic resistance genes but keep pDummy.
3.2 Development of addiction – use of the lambda RED recombination system
To develop the addiction in the host bacterium to pDummy, an essential gene for survival is to be deleted from the bacteria genome, provided that the bacteria can survive on extra-chromosomal copies after the deletion.
The deletion here is mediated through the lambda RED recombination system
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Nat’s part
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The lambda RED recombination cassette is located on the pToolkit (and hence the name of the plasmid). Once the recombination is successful, it can be eliminated from the host bacterium together with the antibiotic resistance gene.
Therefore, once the co-transformation of pDummy and pToolkit is successful, linear dsDNAs having a reporter gene flanked by homologous sequences to the essential gene can be introduced into the bacteria.
When the recombination is kicked started, the essential gene will be swapped out and the reporter gene will be incorporated into the bacteria genome.
Since the linear dsDNAs do not have origin of replications, they are not inherited in daughters unless they are swapped into the genomes. Thus, any observable signals from the reporter would allow identification of successful recombination. Identified colonies can then be further treated to induce loss of pToolkit, which afterwards would be the completed strain of EX.
3.3 Complementation between reporter genes – manifesting completion of EX engineering
To ensure that the final strain of EX has: 1. successfully had its essential gene deleted from genome, 2. maintained the pDummy, a complementation reporter system between the pDummy and swapped gene is preferred over a single reporter at the swapped site.
Different methods can achieve the above aim:
i. Alpha complementation can be used in E. Coli strains where the lacZ gene is completely removed. The larger fragment ω can be swapped for the essential gene while the smaller α fragment can stay on pDummy. In a X-gal rich medium, blue colonies suggest the desired engineered strains.
ii. Complementation between split fluorescent proteins (sFP). 2010 iGEM Slovenia team has demonstrated the principle that N-terminal and C-terminal fragments of sFPS are able to complement in vivo and two sets of sfFPS are able to undergo Forster resonance energy transfer (FRET). This idea is adopted but an alternative set of candidate, split superfolder GFPs (sfGFP), was developed.
3.4 Summary of construction flow:
1. Assembly pDummy and pToolkit
2. Co-transform both plasmid into E Coli and maintain stable strains
3. Introduce linear dsDNAs and induce recombination
4. Isolate recombinants
5. Induce loss of pToolkit
