Why Do We Need Maxicells?

Bacteria and other unicellular prokaryotes may seem somewhat insignificant in the ecosystems we see around us. How can something so small have any influence on the hugely complex multicellular life sharing the environment. We (think we) know all about the bacteria involved in the nitrogen cycle, carbon cycle - bacterial species that we know have a direct affect on the anthropogenic sphere. But what about microbial species that are not directly involved in these processes, should we be concerned about the effects that human activity have on them if it will have no effect on us? The answer is yes. Recent studies in metagenomics have revealed a complex network of dynamic interactions in microbial communities. Species that were previously thought to be unimportant have been found to impact these processes by providing services to those species performing the key, central reactions. With this knowledge it is clear that any human activity, such as environmental release of reproductive micro-organisms, which interferes with the environmental microbiome can have unknown, possibly long-lasting effects on the biogeochemical cycles which are necessary for life on Earth. It is therefore imperative that any Synthetic Biology venture intended for environmental release should not have any undesired effects on the local ecology.

We considered the possible reasons for why environmental release is so dangerous, and decided that there were two main problems which must be overcome:

A series of ‘what ifs’. So we have an instructor plasmid in our maxicells. The maxicell has been released into the environment. How do we prevent our maxicells, and the genetic material they contain, from influencing the finely balanced dynamics in a natural bacterial community, other than the carefully planned effect that is intended?

Maxicell Protocols

Initially we were going to work with Minicells - achromosomal cells generated by the misplacement of the FtsZ ring during replication. However minicells presented problems with purification of the minicell culture due to inconsistent sizing of the minicells and and their tiny size. Maxicells presented as a more useful alternative and easier to purify. We investigated 3 ways in which to produce maxicells in order to present the easiest, most efficient production method for current and future synthetic biologists wishing to use them as a chassis!

Colicin Kill Switch

As the first ‘lock’ in our triple lock system, the function of the colicin kill switch is to prevent the instructor plasmid from being released from the maxicell into the environment in the first place. This 2-part system was inspired by Darmstadt 2016 iGEM team, but altered and optimised for use in maxicells.

The first alteration was to the Immunity protein construct (Imm2). We have inserted ISce-1 sites flanking the Imm2 gene in the plasmid. This makes the system compatible with the ‘Homing Endonuclease’ (ISce-1) method of maxicell production. When ISce-1 expression is induced to digest the chromosome, the Imm2 gene will also be cut out and therefore is no longer expressed. Imm2 protein levels will fall over time until Colicin protein is no longer bound by Imm2 and is free to digest the plasmid.

Following environmental release, conditions for the maxicell will be uncontrolled and variable. Therefore, the longer the maxicell is in the environment, the higher the probability of a chance maxicell lysis event, and release of the plasmid into the surroundings. The plasmid may then be picked up by other prokaryotes in the environment. It is important that the instructor plasmid is degraded before this occurs. However, we do not want to inhibit maxicell function (whatever that may be) by destroying the instructor plasmid too early. Ideally, plasmid degradation should occur immediately prior the to the end of the active metabolic lifetime of the maxicell. Timing of plasmid destruction by colicin should be finely tuned meaning selection of promoters and terminators for Colicin and Imm2 expression is crucial in order for degradation to occur at the desired point in time.

We have used rational design in order to select the promoters and terminators necessary for triggering plasmid degradation at the point we estimate our maxicells will no longer be metabolically active (see ‘active metabolic timeframe’ section). We have designed a model that predicts the degradation time point for every possible promoter-terminator combination for Colicin and Imm2 using the promoters and terminators on the iGEM registry. From this model we selected [promotor x] and [terminator x] for colicin, and [promotor Y] and [terminator Y] for Imm2.

Semantic Containment

The second lock in our triple lock system ensures that even if the first lock fails, an environmental cell would not be able to express and utilise a gene obtained from our maxicells. The basis for this came from Paris Bettencourt 2012 project but we have taken it a step further. Before designing parts for this, we again approached the problem by utilising rational design. We modelled the probability of a translational read-through with increasing numbers of amber stop codons present in a coding sequence. With 2 amber codons, 1 in every 10,000 cells in a culture will be able to generate a read through and express the recoded gene [https://2018.igem.org/Team:Edinburgh_UG/Semantic_Containment_Modelling]. When aiming for complete safety and containment, this is not good enough… We therefore designed 2 new parts building on Paris Bettencourt’s: P1003 5* [BBa_K2725012] and P1003 10* [BBa_K2725013]. These have 5 and 10 serine codons from the P1003 coding sequence substituted with amber stop codons. With 5 amber codons present in a coding sequence, probability of a read through and subsequent expression is reduced to 1 in every 1010 cells, and with 10 amber codons this is reduced further to 1 in 1018 cells [https://2018.igem.org/Team:Edinburgh_UG/Semantic_Containment_Modelling].

Addition of more amber codons however, also presents a problem for expression of the recoded gene in our chassis. In order to solve this we used the same probability model in order to maximise read through in our chassis. We took the supD + terminator sequence from Paris Bettencourt's part, BBa_K914000 and assembled this downstream of a higher strength constitutive anderson promoter. Higher levels of amber suppressor supD increases the likelihood of the tRNA interacting with an amber codon instead of Release Factor 1 that would release the nascent polypeptide from the ribosome and halt translation. We designed 3 of these parts with varying strength anderson promoters in order to test for proof of concept:

• J23102-supD - [BBa_K2725014] (strength 0.82)
• J23103-supD - [BBa_K2725015] (strength 0.01)
• J23108-supD - [BBa_K2725016] (strength 0.51)

Triclosan

The third lock in our triple lock system ensures that even if an environmental cell takes up an instructor plasmid, and can read and express the genes present, that those genes confer no competitive advantage to the environmental cell. This is done by replacing the antibiotic resistance gene in the standard iGEM backbone with a gene that gives resistance to the biocide, triclosan. Triclosan is a chemical that has previously been used in health and beauty products such as toothpaste, but today it is very rarely used. Critically, it is not used in medicine and healthcare, therefore if a pathogen became triclosan resistant this would not interfere in our ability to treat such an infection. Additionally, the triclosan resistance gene, FabV, offers no cross resistance to any commonly used antibiotics.

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