1.1. Theory – how to select?
The E. trojan is a synthetic E.coli strain that is engineered to lack an essential gene, nadE, in its genomic DNA. To survive, this strain has to rely on a pre- introduced plasmid (pDummy) bearing the essential gene; thus forcing the bacteria to maintain the plasmid until an alternative source of nadE gene is present. The pDummy, however, has been designed to have a temperature- sensitive origin of replication which would cease to function if the bacterial cells are incubated under higher incubation temperatures (>42ᵒC???).
For sub-cloning purposes, an E. trojan – compatible vector plasmid is designed. This carrier vector, like the pDummy, contains the nadE essential gene. Once a gene of interest is inserted into this vector, the plasmid can be transformed to the E. trojan for amplification. Incubating the transformed bacteria at a temperature high enough to inactivate the heat sensitive replication origin of the pDummy would result in pDummy loss, making it necessary for the cells to retain the insert- bearing pCarrier for survival. Bacterial cells that do not take up the pCarrier and its insert would be deprived of the nadE gene product and die; while those who do would survive and continue dividing.
1.2. Method of assembly
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 bacteria strain that can perform plasmid selection without the use of antibiotics, and contains as little antibiotics resistance gene as possible.
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 extragenomic 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 (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.
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