CELLECT consists of independent individual parts that have defined functions in nature. By combining these and allowing them to work together, we created a system with new functionality.

The three parts of our foundational system are: a toxin-antitoxin System (TAS), a riboswitch and the gene for production of a compound.

The functionality of each individual part first needs to be tested separately to eliminate the risk of using defect parts to create our system, thereby abolishing a possible source of error when combining the elements with each other.

Knowing we would be working mainly with E. coli we decided to build our system out of parts that are native to E. coli. Their existence and functionality in E. coli reduces the risk of using faulty parts. If you are interested in learning more about our parts, check out our parts page for more details.

The Detector - A Riboswitch

To detect our produced compound we decided on using a riboswitch, a natural mRNA gene-regulatory element that undergoes a conformational change in structure once the target molecule binds. This conformational change regulates expression of downstream genes by various mechanisms. Only two of these mechanisms are important for the riboswitches we selected [1].

(A) First, on the transcriptional level, an intrinsic terminator stem is formed once the ligand binds to the aptamer domain of the riboswitch. The riboswitch controls transcriptional repression. It has a switching sequence that directs the formation of a Rho-independent transcriptional terminator, a short stem-loop structure followed by six or more uridine residues, which signals RNA polymerase to abort transcription. This termination of transcription occurs after the coding part has been transcribed.

(B) Secondly, on the translational level the conformational change of the riboswitch, caused by binding the ligand, hides the access to the ribosome binding site (RBS). The ribosome is unable to form a stable complex with the mRNA and translation initiation is inhibited.

For the proof of principle of our system we needed a compound which is not only detectable with a known riboswitch but also producible in E. coli by introducing as few genes as possible. During our research, we came across multiple interesting compounds. The compound we finally settled on is adenosylcobalamin, also known as vitamin B12. Adonosylcobalamin is the metabolite with the most widespread class of riboswitches in bacteria [2].

We chose three different riboswitches to test and later on decided which would be used in our final system.

The first riboswitch we decided to work with originates from E. coli K12, more precisely in the 5´-UTR of the btuB gene [3]. This riboswitch will be referred to as K12 riboswitch. In nature, this riboswitch regulates the transporter protein BtuB, which is responsible for transporting adenosylcobalamin into the cell. As soon as adenosylcobalamin is present in sufficient amounts, transcription and translation of the transporter gene are stopped by this riboswitch.

The second riboswitch originates from Salmonella typhimurium/enterica [4] and is very similar in structure to the K12 riboswitch. It will be referred to as SY riboswitch. Both the K12 and the SY riboswitch work in the same way by regulating the bluB gene.

Lastly, as we also plan on establishing our system in a more practical chassis organism for bioproduction, cyanobacteria, we also needed a riboswitch capable of differentiating between varying derivatives of our compound of interest, as wild type cyanobacteria are producing pseudo cobalamin naturally. Therefore, the third riboswitch is the PF riboswitch, which originates from Propionibacterium Freudenreichii. It is native in the 5´UTR of cbiB gene [4].

To test the functionality of the different riboswitch variants, we developed a biosensor that indicates the presence of AdoCbl by fluorescence. Since the riboswitch inhibits transcription/translation of downstream genes upon binding to AdoCbl, we would detect a decrease of fluorescent signal if we simply combined the riboswitch with a fluorescent protein. For us, this did not seem like the best solution for a readout. Inspired by the work of Yingying Cai et. al. [3], our solution is to control the expression of the LacI repressor by the riboswitch (Figure 2). With this, we can produce a positive readout for the activity of the riboswitch. Presence of AdoCbl results in an increased expression of a fluorescent protein (e.g. superfolder GFP)

This way, we developed a possibility to test different riboswitches and explore their specificity towards AdoCbl, as well as a detection method for AdoCbl. More details on the detection of our compound of interest, is available on our Sensing B12 Results page.

The Regulator - Toxin-Antitoxin Systems

Toxin-Antitoxin systems (TAS) are widespread among prokaryotes and frequently present in numerous copies in an organism. TAS ensure only the survival of the bacteria carrying mobile genetic elements (such as plasmids) by selectively eliminating the cells that have lost them: if the plasmid is missing, the unstable antitoxin is degraded first and the remaining stable toxin eventually kills the cell.

Looking into multiple options for TAS we noticed that mazE/mazF is not only one of the broadest studied and best documented known TAS, but it has already been used as part in the iGEM competition in previous years [5,6]. The toxin, MazF, is an endoribonuclease: unless neutralised by the antitoxin, MazE, the toxin inhibits translation by cutting cellular mRNA which results in growth arrest and eventually leads to cell death. MazE/MazF is also natively present in E. coli where it serves, among other roles, to combat phage infections in bacterial populations [7].

The toxin-antitoxin complex which neutralises the toxin, is a heterohexamer, with two antitoxin proteins binding four toxins. So simplified one antitoxin protein neutralises two toxin molecules [Figure 4][8].

In CELLECT the expression of the toxin gene mazF will be controlled and negatively regulated by the chosen riboswitch. When adenosylcobalamin is present in the cell, the riboswitch inhibits toxin expression. In the absence of B12, the riboswitch remains inactive and toxin is expressed, eventually leading to cell death. If you want to know more about our work with TAS have a look at our Toxin-Antitoxin Results page.

The Product - B12/Adenosylcobalamin

We choose vitamin B12 as the target compound for multiple reasons, one of them being its relevance for public health, particularly in populations with low or no intake of animal-origin foods.

Vitamin B12 is a crucial nutrient that plays a fundamental role in various bodily functions. It is essential for the production of red blood cells, the maintenance of a healthy nervous system, and the synthesis of DNA. Alarmingly, between 2.5% and 26% of the general population experience B12 deficiency [9]: it affects individuals across all age groups, with a particular emphasis on the elderly population. Moreover, infants, children, adolescents, and women of reproductive age face a higher risk of deficiency, especially in populations which have limited access to or avoid B12-containing animal-derived foods. While animal foods are considered the main dietary sources of vitamin B12 [10], it is worth noting that it is bacteria that synthesise vitamin B12, which is then concentrated in the bodies of higher organisms - mammals cannot produce vitamin B12 themselves.

To produce Adenosylcobalamin (AdoCbl), a specific bioavailable form of vitamin B12, from elemental substrates in E. coli, a total of 28 genes would need to be added to the genome of E. coli [11]. This would not have been feasible for our project. Fortunately, E. coli natively contains the salvage pathway which enables the cells to produce AdoCbl with two substrates, cobinamide (Cbi) and 5,6-Dimethylbenzimidazole (DMB). By adding a single gene, bluB, only Cbi is needed as a substrate anymore, as bluB produces DMB out of FMNH2. You can find more about the B12 production on our B12-Production Results page .

Like a Puzzle - Our Cloning Strategy

A key element of synthetic biology, or science in general, is standardisation. It allows for use and possible expansion of the original idea by other researchers. To design our system, we used parts from the iGEM registry and constructed composite parts that are modular and compatible with the registry. To get an overview of all our parts, have a look on our Parts page.

In order to test each part individually and to simplify the cloning process, we decided to use Golden Gate Cloning. This cloning technique is especially practical when multiple plasmid pieces need to be cloned together and should be interchangeable [12]. Our entire cloning journey is accessible on our Cloning page.

Aside from having to be tested individually, the interaction of the three parts requires a lot of fine-tuning so that they fit together and complement each other like puzzle pieces. Only if all parts work together and are expressed in the needed amounts, the system will be functional. Therefore, modeling played an important role in the design of our system. More details about our modeling are accessible on our Modeling page.

Cells are either capable of production or growth. Therefore, to reach optimal production conditions a certain cell density needs to be reached before the production can be induced. This way, it is ensured that enough cells are capable of production. Inducing the culture too early means less cells are present to produce the desired compound, resulting in lower yields.

Logically, we put the gene for the production dependent enzyme bluB under an inducible promoter, TetR, to control when production is started. In our plasmid design, induction of bluB also induces toxin production, as both genes are expressed under the same promoter. It is important that cell death is not induced before the produced adenosylcobalamin is capable of activating the riboswitch and repressing the toxin production, as this would cause premature cell death. As adenosylcobalamin synthesis is a multistep pathway with multiple enzymes involved, the production of our chosen compound will be noticeably slower than the simple toxin expression. This was proven by our models, which you can view on our Modeling page. For this reason, we set the antitoxin mazE under a constitutive promoter, Amp. This way, any produced toxin at the onset of production will immediately be neutralised. Toxin and bluB are under the same promoter because we want CELLECT to function with a single induction step, solidifying the idea of the system being autoregulatory.

Taking all parts together, we developed an autoregulatory system for vitamin B12 production in E. coli that requires only ONE plasmid. This contains one gene necessary for AdoCbl precursor production, an AdoCbl-specific riboswitch and a toxin and antitoxin gene. AdoCbl production inhibits the toxin transcription and translation, and ensures cell survival. If there is a lack of sufficient AdoCbl production (e.g. due to mutation), the toxin levels increase, leading to cell death. Hence, only the productive cells are CELLECTed to survive.

Challenges and Improvements - What happens if the toxin mutates?

The most susceptible sites for mutations in our CELLECT plasmid are the toxin gene mazF and the TetR promoter in front of bluB and mazF. As the cells are exposed to a selection pressure to stop production of the toxin, new subpopulations with mutations of these plasmid parts could emerge .

Firstly we need to check whether the toxin mutates at all. A simple test to check for mutations is to grow bacteria containing the toxin gene on a plasmid for a certain amount of time and send the plasmid for sequencing to check for changes. Results for the first instalment of this experiment are on our Toxin-Antitoxin Results page.

To further improve our system and secure those weak spots, we designed a plasmid which includes a selection marker, in addition to the already present antibiotic resistance, in front of the riboswitch followed by the toxin gene. This way, the selection marker, e. g. an antibiotic resistance or important part of the metabolite pathway, would force the cell to transcribe and translate this part of the plasmid and subsequently protect toxin and promoter from mutations.

As proof of principle for this further developed version of CELLECT, kanamycin resistance was chosen as a selection marker. Have a look at the first test with this improvement on our Composite Results page.

If you are interested in the future of CELLECT and the results we were able to generate so far for our best composite part, visit our Composite Results page.

Universal Application of CELLECT

To go even further, our goal is to establish CELLECT as a universal autoregulatory system that can be used for different products and in different organisms. So besides E. coli, we intend to establish and test the system in cyanobacteria, more specifically, in the model strain Synechococcus elongatus PCC 7942.

The use of cyanobacteria as sustainable microbial biofactories holds immense potential. These organisms can efficiently convert atmospheric CO₂ into valuable products, utilizing light as an energy source [13,14]. However, phenotypic instability remains a major challenge for scale up of cyanobacterial bioproduction [13].

But why start with Synechococcus elongatus PCC 7942 specifically? It is a strain particularly interesting for de novo vitamin B12 biosynthesis because it already contains major parts of the B12 pathway. Cyanobacteria are capable of producing pseudo cobalamin (pseudovitamin B12), but not DMB. By adding the bluB gene, AdoCbl production is possible by allowing the production of DMB. The theoretical foundation for Adenosylcobalamin production in cyanobacteria was provided by UCSC iGEM 2017 team [15] and master´s thesis of the team member McKenna Hicks [16], we decided to explore the possibilities of AdoCbl production with this promising chassis, which, upon our knowledge, hasn´t been done yet. What we have achieved with cyanobacteria you can read on our B12-Production Results page.

References