A great part of the functionality of Hormonic is thanks to the sensing properties. Hereunder is described how an intein-based GFP reporter proportional to the sensed T3 hormone was developed.
How can bacteria sense the T3 hormone?
As explained on the background page, T3 can be a good marker to determine the thyroid hormonal levels of the body . However, sensing requires the marker, but also the receptor. Actually, our body offers a specific subset of receptors for thyroid hormones (Fig.1), the thyroid hormone receptors (THRs). The THRs are a group of nucleus proteins that can bind to a specific hormone thanks to its Ligand Binding Domain (LBD), which interacts with T3 . Once this happens, THR binds to a specific sequence using its DNA Binding Domain (DBD), then it starts recruiting proteins to unpack the chromatin and to induce gene transcription . However, the mechanisms for gene transcription are specific for mammalian cells, and we cannot be sure about their feasibility in bacteria. Then, another approach is needed.
Why can inteins be so helpful?
While searching for alternative solutions to sense T3, we found an interesting type of proteins, the inteins. Inteins are a family of intervening proteins that carry out a post-translational event known as protein splicing: once the mechanism is activated, they are able to remove themselves from the peptide-chain joining the two remaining portions at both sides . Just as a transposon would cleave and remove itself from a DNA strand. The great advantage of this process is that it is spontaneous, that means, neither external factors, not even energy sources (like ATP) are needed to splice the protein .
A study on bacterial T3 biosensing shows that the presence of the hormone can be sensed with E. coli through an engineered protein that contains an intein domain: a thymidylate synthase (TS) reporter enzyme (required for E. coli cell growth) linked to the Mycobacterium tuberculosis (Mtu) RecA intein splicing domains (Fig.4) and a maltose-binding domain. To make the sensor sensible to T3, the homing endonuclease domain from the Endonuclease PI-MtuI from the Mtu RecA protein was substituted by the LBD of the THR-β, only leaving the intein splicing domains from the original Mtu RecA protein. This chimeric protein sequence that coupled its splicing activity with the T3 binding is named ΔI-SMTR (Fig.5). Then, the TS reporter enzyme is only active when splicing of the intein occurs, that is, cells only grow in the presence of T3. The hormone concentration in the media was reported as the proportional cell growth (OD) at a given time .
How can we design a better sensor for our aim?
Since the aim of Hormonic is to couple the biosensor to electronics and, moreover, in a continuous way, the already existing T3 sensing recombinant protein should be redesigned. Coupling a biologic signal to electronics could be achieved by the use of a photodiode so that the end-product of a genetic circuit is a fluorescence reporter sensed by the electronic component. With that, it was considered that a Fluorescence Protein could be a good reporter, as its signal could be sensed by a photodiode connected to the electronic circuit. This coupling of a biological signal to electronics can be widely seen in the Implementation page.
Despite there are several reports on split GFPs with different inteins, fewer reports on the usage of the Mtu RecA intein with GFP are available . Gangopadhyay et al. study  shows that the use of GFP linked to the Mtu RecA splicing regions ends up forming inclusion bodies, which block the fluorescent signal that the GFP should be emitting. To solve that, they add the GFP to one side of the intein and a maltose-binding site (MBS) acting as a solubility tag to the other one, so that inclusion bodies can be avoided.
Biosensor building parts
Let's build our intein-mediated biosensor!
Let’s now see how we have gathered all the above information and come up with a working T3 biosensor.
First of all, we have to keep in mind that the sensing itself will be produced at a post-translational level, that is, once proteins are already formed. Then, it makes sense to have a gene construct that constitutively produces the protein complex. For that, parts of the iGEM registry were explored, and we ended up using the BBa_K880005 construct, which contains the BBa_J23100 constitutive promoter and the BBa_B0034 ribosome binding site. The combination of both would give a high expression level of the protein complex.
Now, it is time to create the protein complex, which will finally compose the biosensor. When designing this part, we used the Mtu RecA intein previously mentioned, which allows to work with a unique polypeptide chain. However, the optimized ΔI-SMTR sequence published by researchers at the University of Princeton was used, as it proved to have more active splicing domains since it had a replacement of Val67 with Leu (V67L) . In the same way as before, residues 110 to 382 of the optimized Endonuclease PI-MtuI sequence by the LBD of the THR-β resulted in an intein which initiates its activity when T3 is present in the media .
For the extein (the side proteins that the intein will splice and join), as mentioned before, we intended to use GFP to have a luminous signal that can be sensed by an electronic circuit. We made use of state of the art but also of advice from our instructors and it came up that the most reasonable option was using enhanced GFP (eGFP) (BBa_K3484002) split between the residues 70-71 . Later on, we also used the superfolder GFP (sfGFP) (BBa_K3484000), splitted in the same way to compare their activities (between residue 69 and 70). It is important to bear in mind that the decision of the splicing point, aside from allowing an optimal fusion, needs to take into account that the C-terminal of the intein has to be followed by a Cys, Ser or Thr residue .
Furthermore, given the propensity of the intein-GFP complex to form inclusion bodies, as other studies have shown, we also added the solubility tag Fh8 at the end of the N-terminal of the second half of the eGFP and sfGFP, considering its short length and its high solubility power . To end up with the protein complex, we added a flag tag at the C-terminal of the first half of the eGFP and sfGFP to be able to easily characterize the construct by Western-blot at our laboratory.
The sequence needs to end with a terminator, which again was identified from the iGEM registry. The selected one was BBa_B0014 double terminator.
Finally, it was considered the future use of the construct and possible clonings for better expression. That is why biobricks prefixes and suffixes were added at the terminals of the construct (EcoRI and XbaI before the promoter and SpeI and PstI after the terminator).
Accessing the lab under COVID-19 conditions
As until mid-July 2020 we did not have access to the lab, the construct was previously engineered and optimized step-by-step in Benchling’s software so that when being able to access the lab, experiments could immediately start. Then, when access to the lab was confirmed, the construct was ordered to Integrated DNA Technologies (IDT). Further on, the transformations of the sequences to BL21 E. coli strain for expression and to DH5⍺ strain for storage were performed by electroporation and heat-shock, respectively. Once cells were transformed, the intein activity was checked by Western-blot using the BL21 strain, using samples previously grown with and without T3 hormone. Finally, the confirmation of the intein activity led to characterize the biosensor in the plate-reader, proportioning to the cells different concentrations of T3 hormone. With that, fluorescence expression was confirmed and characterized.
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