Edinburgh iGEM 2018

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:

The possibility that our released strain could proliferate uncontrollably
The risk that genes from our strain could escape by horizontal gene transfer and provide a competitive advantage to native microorganisms

Both of these outcomes have to potential to alter the dynamic equilibrium of the microbiome, which could have myriad unforeseen effects.

In order to tackle the first problem, and to a large extent the second, we first looked at minicells: cells generated by abnormal septum formation during replication due to misplacement of the FtsZ ring, leading to the production of small cells that don’t possess a chromosome. As they have no chromosome, they are unable to reproduce and proliferate in the environment. Minicells have been shown to retain plasmids, and are therefore a promising novel chassis for Synthetic Biology: it would be possible for your minicells to perform any plasmid-encoded function you desire, and be safe for environmental release. However, minicells presented problems with purification of the minicell culture due to their small, inconsistent size. After discussion with experts in the field of biotechnology to try and resolve these issues, it was suggested that we instead try to remove the chromosome by some other means. This suggestion led us to completely change the design of our project; to instead look at maxicells. Maxicells are bacterial cells which have lost their chromosome, and are therefore unable to reproduce and proliferate in the environment. These maxicells can be used for Synthetic Biology, as they retain any plasmids they possessed prior to maxicell production, which we have defined as the “instructor plasmids”. Maxicells would then be as functional and safe as minicells, without the costly, time-consuming purification steps.

While maxicells are unable to reproduce and cannot release any chromosomal genes into the environment, it is nonetheless still possible that advantageous genes could escape by horizontal gene transfer from the instructor plasmid, the most notable of which being the antibiotic resistance marker used for plasmid selection. We have approached this problem in three ways, which we have termed our Triple Lock System.

The first lock in our Triple Lock System is the colicin kill switch, which aims to fully degrade any plasmid DNA in the maxicell after a defined, programmable time-frame. The second lock is our semantic containment system, which ensures that any native microorganisms will be unable to read the instructor plasmid DNA, as it will be written in a different language. The final lock is the alternative selection marker, triclosan, which removes the need for an antibiotic resistance gene to be encoded on the instructor plasmid, thereby preventing the spread of antibiotic resistance.

The design of our project then fell into five overarching sections, which will be detailed below:

Evaluating and Optimising Maxicell Protocols
Quantifying the Active Timeframe of Maxicells
The Colicin Kill Switch
Semantic Containment
Alternative Selection

Evaluating and Optimising Maxicell Protocols

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! These three methods were by: ultraviolet light exposure; cleaving a palindromic hairpin loop on the chromosome; cutting the chromosome with a homing endonuclease. We then attempted to modularise the UV production method by creating knockdowns for DNA repair genes using RNA interference.

UV Production Method

This is the established method of maxicell production, which uses ultraviolet light to damage the DNA within the cell. This causes pyrimidine dimers to form, which would normally be restored by nucleotide excision repair. However, the maxicell producing strain is a knockout for recA - an enzyme involved in DNA repair by homologous recombination - and uvrA - an enzyme involved in nucleotide excision repair.

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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