Design

In our biosensor, the pSynSense2.5000 and NarX-NarL two-component system are used. pSynSense2.5000 is a plasmid with a pSC101 backbone and biosensor circuit developed by UGent (De Paepe et al., 2018). The circuit is composed of a detector and effector module controlled by PfdeAR-FdeR (P_Naringenin), which is a naringenin-responsive promotor (Figure 1).

Firstly, NarX, P_NarL, and mKATE are inserted downstream of the effector module. The therapeutic protein downstream of P_NarL is replaced by a red fluorescent protein, mKATE, to quantify the expression (Figure 2). Two parameters can control the expression of mKATE which will be measured using a microplate reader. Different concentrations of naringenin (0, 20, 40, 60, 80, 100 mg/L) show the dose dependency of the NarX expression, while presence or absence of nitrate will control the dimerization and phosphorylation of NarX.

The second construct consists of the same genes except for the NarX wildtype which is replaced by the mutant (Figure 3). Histidine 399 of NarX is replaced by glutamic acid which allows NarX mutant to dimerize, but blocks the phosphorylation of NarL (Cavicchioli et al., 1995). All experiments done for the wildtype NarX are done for the mutant. However, there should be no mKATE expression as NarL will not be phosphorylated, resulting in no expression of P_NarL and its downstream genes. In addition, SDS-PAGE is performed to verify that NarX mutant is still expressed, and the mKATE is not absent because of the misfolding or absence of NarX mutant.

Finally, the third construct, NarX mutant under the control of the tetracycline-inducible promoter is inserted in another compatible plasmid. pSynSense2.5000 is a plasmid with a pSC101 backbone, thus the NarX mutant is inserted in pLO_SNAP. The Tet promotor ensures controllable constant expression of NarX mutant. This plasmid is co-transformed with the first construct. NarX mutant will quench the wild-type phosphorylation. Thus, it is expected that mKATE expression is lower than in the first construct until it passes a certain threshold of naringenin concentration.

NarX wildtype and mutant are both fused with two different fluorescent proteins to observe their binding in the membrane and interaction between each other. Wildtype NarX is fused with mNeonGreen, thus successful cloning will show a green membrane indicating the presence of NarX. On the other hand, mutant NarX is fused with a yellow fluorescence protein, mPapaya. The interaction of NarX wildtype and mutant (Figure 5) is observable using Fluorescence Resonance Energy Transfer (FRET). The fluorescence pairs were chosen using the FP base. NarX-mNeonGreen acts as the donor while NarX mutant-mPapaya acts as the acceptor. There will be three interactions observed in the co-transformants showing different fluorescence:

• NarX wildtype-wildtype interaction
• NarX wildtype-mutant interaction
• NarX mutant-mutant interaction

Engineering cycle and success

After obtaining the plasmid from professor De Mey’s research group at Ghent University (Belgium), a colony from the chloramphenicol selection plate was picked and grown in liquid medium to prepare for plasmid extraction. After plasmid extraction the following day, the plasmid was fully sequenced as suggested by Dr. De Paepe. When the results came out positive, the first iterations of plasmid construction could then begin.

The pLO_SNAP plasmid was amplified in such way that OmpA, the SNAP-tag and the Lpp signal peptide were removed, consequently the pSynSense2.5000 was amplified to retain the part of the detector and effector modules needed for our design. The amplification of the pSynSense2.5000 plasmid was repeated due to a low yield, the primer annealing temperature was lowered to 64°C instead of 72°C which was too high as an extension temperature. After successful amplification of the plasmids, the gBlocks were amplified. All results were as expected, which was shown with an agarose gel electrophoresis (Figure 6-7). All PCR products were purified from the reaction mixture with the E.N.Z.A.® Plasmid DNA Mini Kit I (Omega Bio-tek, Georgia, USA).

Next, all PCR products were digested with SapI and the following products were combined in a ligation mixture: pLO and NarX mutant – mPapaya (3:1 ratio), NarX – mNeonGreen and P_NarL mKate (1:3 ratio), NarX mutant – mPapaya and P_NarL mKate (1:3 ratio). The ligations of those parts were checked by digesting an aliquot with EcoRV-HF and the result was visualised using agarose gel electrophoresis but no bands showed. Therefore, the ligation was performed again but the method was changed. The following ligation mixtures were set up with the PCR products of the original plasmids and the gBlocks: NarX – mNeonGreen with P_NarL mKate and pSynSense2.5000, NarX mutant – mPapaya with P_NarL mKate and pSynSense2.5000. With the previous ligation products, the following reactions were set up: NarX – mNeonGreen – P_NarL mKate and pSynSense2.5000, NarX mutant – mPapaya – P_NarL mKate and pSynSense2.5000. After incubation at the appropriate temperature for the appropriate time, the results were directly tested by transforming into chemically competent E. coli DH5α cells.

After a second try of this last described method, colonies grew on the selection plates where E. coli DH5α cells were plated containing the following constructs: pSynSense2.5000_NarX_mKate (1 and 2 step ligation), pSynSense2.5000_NarXmutant_mKate (1 and 2 step ligation). Another round of pLO_NarX mutant-mPapaya plasmid construction was performed but no colonies grew on the selection plate which contradicted the agarose gel electrophoresis (Figure 8-13). Although the colony PCR shows a negative result, the linearized plasmid shows the correct size fragment. Therfore, this pLO_NarX mutant-mPapaya plasmid was sequenced together with the positive hits found through colony PCR (Sanger Sequencing, Eurofins Genomics). The results and subsequent actions are summarized in table 1.

Colony 8 and 30 (Table 1) were incubated in the correct selection medium and a glycerol stock was prepared, while new primers were designed for the pLO plasmid. Another 2 cycles of amplification, digestion, ligation (pLO + NarX-mutant mPapaya) and transformation was performed but the single colony that grew did not show positive results after colony PCR. During every cycle new primers were used, PCR conditions adapted and ligation mixtures were optimized.

The next step in constructing the plasmids pSynSense2.5000 – NarX mNeonGreen – P_NarL mKate, is to open up the pSynSense2.5000 - P_NarL mKate plasmid and ligate the gBlock NarX mNeonGreen into it. The analogous reasoning goes for the plasmid pSynSense2.5000 – NarX mutant mPapaya – P_NarL mKate. This step also required multiple rounds of amplification, digestion, ligation, transformation, colony PCR and troubleshooting but the cloning of this last step was not completed. Transformation in this last step was also attempted in E. coli K12 MG1665.

Future perspectives

To obtain the designed plasmids, there are some strategies possible. A first step would be to set up multiple ligation reactions in parallel where the insert:backbone ratios, the reaction time and temperature are optimised. If the type IIS cloning protocol does not work, different common strategies can be used such as Gibson assembly.

After successful cloning and transformation of the plasmids, the three separate constructs will be tested; pLO_NarX mutant mPapaya, pSynsense2.5000_NarX mNeonGreen_P_NarL mKate and pSynsense2.5000_NarX mutant mPapaya_P_NarL mKate. As described in the design section, the expression of mKate in the pSynsense2.5000_NarX mNeonGreen_P_NarL mKate plasmid is controlled by different concentrations of naringenin and the absence or presence of nitrate. In order to compare these different expressions, different test conditions are used (0, 20, 40, 60, 80, 100 mg/L naringenin, with and without nitrate). The expression of mKate can then be followed with a microplate reader and microscopy.

Following this, the same experiments are performed with the pSynsense2.5000_NarX mutant mPapaya_P_NarL mKate plasmid but no fluorescence should be detected since the secondary messenger, NarL, cannot be activated by the NarX mutant.

In case the fluorescence measurements are negative, don’t show a significant difference or don’t match the desired results, there are some tests that can also be performed to trace the problem. First, SDS-PAGE should be used to see whether NarX and NarX mutant are expressed and mKATE does not remain undetected because of misfolding or absence of these proteins. Additionally, NarX and NarX mutant are fused to a fluorescent protein. The expression of the protein on the membrane could thus be analyzed with a microplate reader.

Lastly, the pLO_NarX mutant mPapaya is co-transformed with the pSynsense2.5000_NarX mNeonGreen_P_NarL mKate plasmid. Induction of the NarX mutant mPapaya expression would be done with a constant concentration of anhydrotetracycline. The cells would be subjected to different concentrations of naringenin with and without nitrate and the fluorescence can be measured again with a microplate reader and seen with microscopy.

The idea is to implement a kill switch that is regulated by temperature. At body temperature (37°C), the cell will survive. When the temperature drops below a certain threshold, the cell dies. The kill switch is based on RNA thermometers that respond to temperature shifts between 37°C and 30°C for the ligation of downstream genes. Three thermometers were chosen, based on literature (see Antitoxin): the 5’-untranslated regions (UTR) of prfA, hsp17 and rpoH. The RNA thermometers will be controlling the translation of the antitoxin, whereas the toxin will be constitutively expressed. For the toxin-antitoxin system, a CcdA/CcdB system was chosen. Both the toxin and antitoxin construct are designed according to SEVA guidelines and cloned into the pLO_SNAP plasmid. The choice of the plasmid is mainly driven by its proven cloning efficiency and availability in the PI’s lab. It has an ampicillin resistance marker and a tetracycline repressor gene (TetR).

General preparations

In order to start the experiments, the plasmids pLO_SNAP and pET28 were purified from E. coli DH5α strains. To visualize a plasmid on a gel the plasmid was digested with a restriction enzyme that has only one estriction site in the plasmid. HindIII and NcoI were used for the restriction of pLO and pET28, respectively. Primers are designed with an overhang including three adenines, which allow for polymerase binding, the BsaI recognition site and restriction site, followed by 4 nucleotides. BsaI cuts one basepair downstream of its recognition site and the four nucleotides will form the sticky end. This restriction site allows for sticky end ligation of two digested PCR products. The same restriction enzyme (BsaI) was chosen for all plasmid and inserts.

• BsaI type IIS overhang: 5’ AAAGGTCTCN | NNNN 3’

At the beginning, the PCR of the plasmid pLO_SNAP failed repeatedly. The reason behind this was a palindromic sequence in the primer overhangs that caused self-annealing of the primers. Primers used for the amplification of the plasmid and inserts were redesigned which resolved the problem. Primers were thoroughly checked using the IDT OligoAnalyzerTM Tool. To troubleshoot the PCR reactions, we added 0.5 µL of formamide to the reaction mixture to ensure a higher primer specificity.

The digestion, ligation, and transformation protocol had to be optimized for some experiments in order to keep damage to the DNA or competent cells minimal and achieve successful cloning. For instance, the heat inactivation step after restriction was not performed and digestion reaction mixtures of both the insert and the vector were premixed before purification. The T4 DNA ligase and its buffer were then added to this premixed purified sample. A Gibson assembly method was also tried for some results but did not lead to favorable results.For the transformation, cells were given more time to chill on ice, and incubation was done without shaking. Streaking was done with 100 µL of the 500 µL transformation mixture whereafter the remaining 400 µL was centrifuged and the supernatant discarded. The pellet was resuspended in 100 µL LB and streaked on another selective plate. This generally led to a successful result. After sequencing results of a constructed plasmid turned out to be positive, a glycerol stock was prepared. For more information about the protocols, please take a look at the Experiments page.

Toxin

The toxin, ccdB is under the control of a anhydrotetracycline- (aTc) and IPTG inducible promoter (BBa_K4345022) and fused with a rigid linker to sfGFP. This is to ensure that the expression is controllable in the lab, to minimize leaky expression and to avoid the toxin having an effect on the viability of the bacteria during the cloning phase. When anhydrotetracycline and IPTG are added, they will remove TetR and lacI from the Tet and lacO operon, respectively, and subsequently make the Tet promotor available for the RNA polymerase. When the system is induced with both inducers, transcription can take place. In order for this design to work, the TetR and LacI gene are also part of the plasmid design.

Besides this double controlled promoter, a stopcodon in each ORF was added to the coding sequence of CcdB. Because of the stopcodons in the toxin gene, the downstream linker and sfGFP should not be translated and no fluorescence should be observed after induction with aTc and IPTG. This stopcodon region is flanked by restriction sites and can be removed with SalI to activate the expression of the toxin only when cloning was successful. The goal of this design is, by having these three control elements, to successfully activate the expression of the toxin in a controlled way with minimal leaky expression and extra burden on the cell when in the cloning phase. More information about this composite part can be found in the parts registry with the code BBa_K4345018.

The TetR gene is already part of the pLO_SNAP plasmid, which serves as a backbone for the toxin construct. The LacI gene still had to be cloned into the pLO plasmid. Cloning the LacI gene is the next step after the successful insertion of the Toxin gblock, generating pLO_Toxin. The LacI gene was amplified from pET28. At first, multiple bands showed on the agarose gel due to unspecific primer binding sites. After gel extraction of the band of interest, the extracted samples functioned as new PCR templates for the LacI gene.

After numerous tries to clone the LacI into pLO_SNAP, agarose gel electrophoresis of the colony PCR products showed promising results. However, the sequencing results showed a single point mutation in the startcodon of the LacI gene. Because of time constraints, other parts of the project were prioritized. Nevertheless, an adequate solution was devised. By transforming the pLO_Toxin plasmid into E. coli DH5α Z1, a strain that has the LacI and TetR gene in its genome, it is not necessary to clone the LacI gene into the plasmid. Therefore, it was possible to continue with measuring fluorescence of the pLO_Toxin construct upon induction (figure 14).

Eventually, it would be interesting to test both the antitoxin and toxin construct in the same cell. The problem is that the pLO plasmid is not compatible with itself. This means that, after the transformation of two plasmids in the same cell, the bacteria are prone to lose one of them. This is especially the case when both plasmids have the same antibiotic resistance marker, because the cell would only need one of the plasmids. Because of this, the toxin is also cloned into another plasmid called pBAD which is compatible with pLO. Also, pBAD has kanamycin as an antibiotic resistance marker. A visualization of the assembled pBAD plasmid can be seen in figure 15.

Fluorescence measurement

As mentioned in the experiments, different types and concentrations of inducer are tested. There is a significant difference between the samples with the negative control indicating the presence of fluorescence. The negative control consists of pLO_SNAP, cloned into E. coli DH5α cells. The fluorescence increases in parallel to the increasing concentration of aTc. However, there is no significant difference between the different concentrations of IPTG (figure 16).

When induced using two inducers (figure 17), pLO_Toxin samples don’t show any fluorescence, while pBAD_Toxin shows a significantly higher fluorescence compared to the control. However, there is no significant difference between the treatments. According to the result, we suspect that the toxin in pLO is expressed, hence killing the cell which causes the reduced fluorescence. This explains why fluorescence is still detected when only one inducer is added. Meanwhile, pBAD_Toxin depicts how high the toxin is expressed with the combined induction with of IPTG and aTc, compared to a single inducer in the previous graph. Because the induction test is only done once, it has to be repeated and carried out in other conditions to generate more data points in order to draw conclusions.

Antitoxin

The thermoregulated transcriptional activator prfA induces the expression of virulence genes in Listeria monocytes (Johansson et al., 2002). It is maximally expressed at 37°C but at 30°C the expression is silent. In addition, rpoH encodes a heat shock σ-factor in E. coli and its regulatory RNA thermosensor unfolds at 30°C (Morita et al., 1999). Lastly, the expression of hsp17 in Synechocystis is regulated by an exceptionally short 5’ UTR which blocks translation at 34°C (Kortmann et al., 2011).

The antitoxin CcdA is under the control of a constitutive promoter and one of the RNA thermometers. CcdA is fused to sfGFP with a rigid linker to follow the expression by measuring fluorescence. The final goal of this design is to follow the expression of the antitoxin at different temperatures. The mechanism of the RNA thermometers is described schematically in figure 6. At body temperature (37°C), the RNA thermometer will unfold and linearise. As a consequence, the ribosome is able to find the ribosomal binding site (RBS) and initiate translation of the downstream antitoxin gene fused to sfGFP. When the temperature drops below a certain threshold, a secondary structure is formed that hides the RBS thus preventing downstream translation (Figure 18).

The RNA thermometers hsp17, prfA and rpoH are registered under BBa_K4534001, BBa_K4345010 and BBa_K4345019 respectively. The composite parts are registered under BBa_K4534020, BBa_K4534023, BBa_K4534024, respectively. The overview of an assembled plasmid with all the components in the antitoxin design can be seen in figure 19. As an example, pLO_hsp17 is shown.

Hsp17 RNA thermometer construct

A visual representation of the secondary structure formed in the RNA sequence of the hsp17 thermometer can be seen in figure 20 and the ordered gBlock in figure 21. Note that the secondary structure is only formed below the threshold temperature, for example at 25 °C. In this state, the RBS is hidden from the ribosome. In this specific RNA thermometer, the start codon is also part of the secondary structure.

After digestion, ligation and transformation, the construct was sequenced and the results turned out positive. Three specific colonies were chosen for fluorescent measurement. However, when fluorescence measurements were taken, the three colonies did not show the same results. These colonies were incubated again from the glycerol stock, whereafter the plasmids were purified and sequenced. As expected, the two deviating colonies showed a deletion of the promoter, RNA thermometer and CcdA gene, as showed in figure 22.

rpoH RNA thermometer construct

The RNA thermometer rpoH has the same design as hsp17. For both constructs, the same protocol was used and experiments were carried out in parallel. The composition of the gblock is showed in figure 23.

prfA RNA thermometer construct

It was not possible to order the prfA construct design from IDT as it was for the other RNA thermometers. The reason was the formation of secondary DNA structures. In consequence, we divided it into two and assembled the construct ourselves. IDT could still provide us with a gblock containing the promoter, RBS, prfA thermometer and ccdA sequence as shown in figure 23. The promoter, RBS and prfA thermometer was amplified from this gblock with the primers 018 and 019, while ccdA linked to sfGFP was amplified from the gblock hsp17 with primers 002 and 020 (Hsp17_CcdA-sfGFP). Figure 24 shows the use of the just mentioned primers. Note that the primers 018 and 002 are also used in the amplification of the other gblocks to add the compatible overhang for insertion into pLO. These overhangs all make use of the same restriction enzyme BsaI. Because of this, it should have been possible to do the assembly of the whole prfA construct and the insertion into pLO_SNAP in the same reaction mixture. However, it was not possible to follow this approach which is why both steps were performed subsequently. When PCR of the prfA gblock ligated to Hsp17_CcdA-sfGFP was done prior to insertion into pLO_SNAP, a higher concentration of insert product was obtained. As a result, it was easier to control the ratio of insert and vector when assembling the pLO_prfA plasmid with our ligation protocol. After the transformation of pLO_prfA, sequencing results turned out to be positive. There can be followed by with fluorescence testing.

Fluorescence measurement

Initially, we wanted to use M9 minimal media to test fluorescence, since it would be less intrusive when measurements are taken. However, we were unable to grow our bacteria in it. Therefore, we settled for LB with ampicillin as our media.

Prior to performing temperature tests, we could visually see fluorescence activity in one of the hsp17 colonies, specifically colony 2 (hsp17 col2). We could see this on the plate as well as in the LB broth solution when grown at higher temperatures (~35°C and higher). An example can be seen in figure 26. At this point we had sequenced colonies of various thermometer constructs: rpoH, prfA, hsp17. Because of what we saw on agar plates, we believed that hsp17 col2 would be the most promising. Moreover, when tested at 25°C and 37°C, fluorescence is only present at 37°C.

Fluorescence was measured with the CLARIOStar. Since the CcdA linked to sfGFP sequence should be expressed constitutively at certain temperatures, we do not need to add an inducer to read fluorescence. Resulting fluorescence expression should ideally be driven purely by the behaviour of the RNA thermometer.

Initially we had tested a few temperatures, but decided that if we controlled more variables we may be able to compare results between temperatures more effectively. We controlled for growth period by incubating wells for 16 hours. Since time was limited, we chose to focus on testing a variety of temperatures, rather than testing multiple times. We were able to collect temperatures ranging from 25°C to 42°C. The results can be seen in figure 28.

We decided to test the hsp17 construct in two additional strains: E. coli MG1665 and BL21. We measured sfGFP expression in these strains at temperatures between 20°C and 37°. We decided to investigate lower temperatures because strong fluorescence could already be seen at 37°C and we are more interested in the ceased expression of ccdA. The results of these measurements are shown in figure 29. Plate images of temperatures 31°C, 34°C, and 37°C incubations are shown (Figure 30) where decreasing fluorescence is seen with decreasing temperature.

When processing the data, we had a wide range of OD₆₀₀ and fluorescence values. To effectively use Fluorescence values of cell cultures, we needed to select readings that had OD₆₀₀ values within a certain range. Ideally all the OD₆₀₀ values should be around 0.5, however, if we were to select only values that are 0.5 ± 0.1 we would end up losing a large part of the data. Considering that, we selected OD₆₀₀ values between 0.4 – 0.6, though we would end up with the same problem. Therefore, we decided to expand our range to 0.2 - 0.8. Although this makes our results less robust, it allows us to have enough data to make general statements about our results.

In order to compare fluorescence between samples, we must normalize for OD₆₀₀. To do this, output fluorescence value of the CLARIOStar reading is divided by OD₆₀₀. The resulting value is what is used in figures 28 and 29 which we refer to as fluorescence/OD₆₀₀. A blank sample of LB medium was also implemented in the well plate, for which the software of the CLARIOstar made the correction. To account for autofluorescence, we could have subtracted the fluorescence/OD value of the negative control (pLO_SNAP, cloned into E. coli DH5α) from relevant samples, however visually this would at times reduce the fluorescence/OD₆₀₀ of samples down to 0 or negative values (which are then set to 0). So, to visualize the results more easily, we decided to forgo the extra calculation and show the autofluorescence on the bar chart itself.

In figure 30 we can see that as temperature increases, fluorescence increases both in strains BL21 and MG1665. This would indicate that our temperature dependent riboswitch hsp17 does work, however expression is not as refined as we would hope. Ideally, for our purposes, we would want a more binary switch on expression. In figure 28, we can see that the other thermometers caused an increase in fluorescence as temperature increased, but none to the degree of hsp17 colony 2.

Furture perspectives

When designing an RNA thermometer, it is possible to integrate part of the downstream gene of interest into the secondary structure of the RNA thermometer. By doing this, the start codon can also be hidden, minimizing leaky expression for more reproducible results. It is also possible to insert specific mutations in the RNA thermometer sequence. As a result, the secondary structure slightly changes, making it less stable so that the RNA thermometer will unfold and linearize at a lower temperature. By changing these parameters, the characteristics of RNA thermometers can be altered to suit your preferences.

In order to make stronger statements about the nature of our thermometers, the next steps would be to test each temperature condition multiple times. We were only able to test conditions at most twice. This results in a very low sample size, making our results unreliable. We believe that much of the variation we see would disappear once a larger sample size is studied. Furthermore, in order to make the most of our data, we should alter our incubation conditions slightly. At first, we wanted to control and standardize our incubation conditions, part of which was being consistent on incubation time. However, it would have been better to monitor the growth of each test condition and take fluorescence measurements once an OD₆₀₀ of 0.5 was reached. Because of how fluorescence readings are taken, values too low or too high can introduce error and make the reading itself unreliable, not to mention the normalized reading. By taking measurements when an OD₆₀₀ of 0.5 is reached, we could better control for these sources of error. We noticed that, sensibly, when incubations were done at higher temperatures, OD₆₀₀ would generally be higher after 16 hours and the opposite is true for lower temperatures. This made comparing results very difficult. Additionally , it would be interesting to test a larger range of temperatures, as well as more temperatures within the range we had. If enough readings were taken, a robust expression curve could be attained that would well describe how the thermometers behave at varying temperatures. We could also look at our results in a different way. Rather than subtracting the autorfluorescence of cells, what may be more telling is to divide by the autofluorescence. This would give us a factor value of fluorescence that may be more indicative of gene expression. Since with absolute numbers, values can only be generally understood, small differences don't reliably mean anything. In conjuction with a cutoff range, a factor of fluorescence approach could be worth doing.

To achieve more comparable results, a positive control should be implemented. For example, plasmid DNA can be labeled with a fluorophore called FITC. The fluorescence of FITC would then correspond to the quantity of plasmid DNA. Next to this, another fluorophore with no spectral overlap with FITC can be used to quantify protein expression. Since the emission wavelength of sfGFP and FITC are both in the green spectrum, it would be best to substitute the sfGFP protein in our design for another fluorescent protein such as mCherry (Homann et al., 2017).

Another interesting experiment would be to follow the development of a fluorescent signal when the temperature changes over time. Our design is capable to be tested in an environment where the temperature is gradually increased over time. In order to do the same with decreasing temperature, sfGFP should be replaced with a fast-degrading variant of GFP or fused to a protease-degradation tag. Note that for these experiments specific equipment is needed.

If two compatible plasmids containing either the Toxin or the Antitoxin are transformed into the same bacterial cell, the functioning of the CcdA/CcdB system can be investigated. In order to do this, it would be best to exclude the linker and sfGFP from the design for the reason that the distinction of which gene is actually transcribed cannot be made after translation. Moreover, it would be of interest to eliminate the possibility that the big fluorescent protein linked to CcdA and CcdB would interfere with their activity because of steric hindrance. By doing so, the cell viability at different temperatures can be followed over time by measuring OD₆₀₀ when the system is induced with anhydrotetracycline and IPTG.

Eventually, when a more thorough characterization in E. coli strains is done, it would be of interest to transfer the project to a Lactobacillus strain because of its probiotic properties. Designwise, some changes have to be made. The following information was provided to us by Prof. Sarah Lebeer through the means of verbal communication. It is often the case that the folding of fluorescent protein GFP in Lactobacillus strains is difficult which is why it would be better to use mCherry as an alternative. Furthermore, the toxin-antitoxin system CcdA/CcdB could possibly not work in Lactobacillus strains because of its specific mechanism of action. An alternative would be the Barnase-Barstar system. The same research group also advised us to use the specific Lacticaseibacillus rhamnosus LMG 18243 strain for cloning. By transferring to the probiotic strain, the project takes on another successive step in its development.