iGEM 2022 — Team ku-leuven

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Notebook

From the very beginning of our labs, we documented our work on the online notebook platform “Benchling” in order to keep track of everyone’s work and stay organized. We divided our notebooks into 4 parts: General Preparations, Toxin, Antitoxin, and Shoot. General preparation is a combination of of both the general preparations notebook and the Toxin/Antitoxin preparations notebook. The Antitoxin notebook is a combination of the prfa ligation notebook, Antitoxin pLO insert notebook, and the construct testing notebook. The notebooks of each section can be found below.

GENERAL PREPARATIONS

If the pdf doesn't show, click [here](https://static.igem.wiki/teams/4345/wiki/notebook-pdfs/general-preparations-compressed.pdf) to download the pdf!

TOXIN

If the pdf doesn't show, click [here](https://static.igem.wiki/teams/4345/wiki/notebook-pdfs/toxin-compressed.pdf) to download it!

ANTITOXIN

If the pdf doesn't show, click [here](https://static.igem.wiki/teams/4345/wiki/notebook-pdfs/antitoxin-compressed.pdf) to download the pdf!

SHOOT

If the pdf doesn't show, click [here](https://static.igem.wiki/teams/4345/wiki/notebook-pdfs/shoot-benchling.pdf) to download the pdf!

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* [Overview](#engy1)
* [EAT - SHOOT](#engy2)
* [LEAVE](#engy3)

# Engineering Cycle: Overview

Our projects covers three parts essential for a biosensor: **Eat**, **Shoot** and **Leave**.

[![](https://static.igem.wiki/teams/4345/wiki/generic/eat.jpg)

EAT

Detect the target in the gut](#engy2) [![](https://static.igem.wiki/teams/4345/wiki/generic/shoot.jpg)

SHOOT

Release of therapeutics](#engy2) [![](https://static.igem.wiki/teams/4345/wiki/generic/leave.png)

LEAVE

Kill-switch for biocontainment](#engy3)

# EAT - SHOOT

#### 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 (PNaringenin), which is a naringenin-responsive promotor (Figure 1).

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture1.jpg)

Figure 1: pSynSense2.5000 PfdeAR-FdeR biosensor circuit (De Paepe *et al.*, 2018)

Firstly, NarX, PNarL, and mKATE are inserted downstream of the effector module. The therapeutic protein downstream of PNarL 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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture2.png)

Figure 2: pSynSense2.5000 NarX wildtype PNarL mKATE

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 PNarL 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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture3.png)

Figure 3: pSynSense2.5000 NarX mutant PNarL mKATE

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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture4.png)

Figure 4: Co-transformation of pSynSense2.5000 NarX PNarL mKATE and pLO NarX mutant

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

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture5.png)

Figure 5: FRET interactions between NarX wildtype and mutant

#### 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).

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture6.jpg)

Figure 6: 20220820 pLO pSynSense (left) and 20220822 pSynSense PCR (right)

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture7.jpg)

Figure 7: 20220829 gBlocks PCR

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 PNarL mKate (1:3 ratio), NarX mutant – mPapaya and PNarL 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 PNarL mKate and pSynSense2.5000, NarX mutant – mPapaya with PNarL mKate and pSynSense2.5000. With the previous ligation products, the following reactions were set up: NarX – mNeonGreen – PNarL mKate and pSynSense2.5000, NarX mutant – mPapaya – PNarL 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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture8.jpg)

Table 1: Sequencing results and trouble shooting

![](https://static.igem.wiki/teams/4345/wiki/engineering/troubleshooting-shoot.png)

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 – PNarL mKate, is to open up the pSynSense2.5000 - PNarL mKate plasmid and ligate the gBlock NarX mNeonGreen into it. The analogous reasoning goes for the plasmid pSynSense2.5000 – NarX mutant mPapaya – PNarL 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\_PNarL mKate and pSynsense2.5000\_NarX mutant mPapaya\_PNarL mKate. As described in the design section, the expression of mKate in the pSynsense2.5000\_NarX mNeonGreen\_PNarL 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\_PNarL 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\_PNarL 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.

# LEAVE

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).

![](https://static.igem.wiki/teams/4345/wiki/engineering/toxin-laci-plasmid.png)

Figure 14: Visualization of assembled pLO\_Toxin and pLO\_Toxin\_LacI plasmids, generated with Snapgene

![](https://static.igem.wiki/teams/4345/wiki/engineering/toxin-plasmid.png)

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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture12.png)

Figure 15: Visualization of assembled pBAD\_Toxin plasmid, generated with Snapgene

#### 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).

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture13.jpg)

Figure 16: Single induction of pLO\_Toxin and pBAD Toxin with increasing concentration of aTC and IPTG

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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture14.jpg)

Figure 17: Combinational induction of pLO\_Toxin and pBAD\_Toxin with increasing concentration of aTC and IPTG

#### 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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture15.png)

Figure 18: Schematic representation of the RNA thermometers. Figure created with Biorender.com

![](https://static.igem.wiki/teams/4345/wiki/engineering/plo-hsp17-plasmid.png)

Figure 19: Assembled plasmid of the Antitoxin construct pLO\_hsp17, generated with Snapgene

#### 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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture17.png)

Figure 20: Structure predicted with UNAfold, adapted in Biorender

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture18.png)

Figure 21: Visualization of the ordered hsp17 gblock design, generated with Snapgene

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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture19.png)

Figure 22: Visualization of pLO\_hsp17 sequencing results in the fluorescent testing phase, generated with Snapgene

#### rpoH RNA thermometer construct

![](https://static.igem.wiki/teams/4345/wiki/engineering/rpoh-snapgene.png)

Figure 23: Visualization of ordered rpoH gblock design, generated with Snapgene

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

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture20.png)

Figure 24: Visualization of prfA gblock ordered from IDT, generated with Snapgene

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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture21.png)

Figure 25: Assembly of prfA gblock and CcdA linked to sfGFP from hsp17 gblock, generated with Snapgene

#### 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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/picture22.jpg)

Figure 26: Preliminary fluorescence test

![](https://static.igem.wiki/teams/4345/wiki/engineering/hsp17.jpg)

Figure 27: Preliminary hsp17 colony 2 fluorescence test

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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/leave/figure-28.png)

Figure 28: Fluorescence by original construct and temperature

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.

![](https://static.igem.wiki/teams/4345/wiki/engineering/leave/figure-29.png)

Figure 29: Hsp17 col2 plasmids in two new strains

![](https://static.igem.wiki/teams/4345/wiki/engineering/leave/figure-30.jpg)

Figure 30: Plate images of hsp17 different temperatures

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.

References

1. Cavicchioli, R., Schröder, I., Schröder, S., Constanti, M., & Gunsalus, R. P. (1995). The NarX and NarQ Sensor-Transmitter Proteins of Escherichia coli Each Require Two Conserved Histidines for Nitrate-Dependent Signal Transduction to NarL. JOURNAL OF BACTERIOLOGY, 177(9), 2416–2424.
2. De Paepe, B., Maertens, J., Vanholme, B., & de Mey, M. (2018). Modularization and Response Curve Engineering of a Naringenin-Responsive Transcriptional Biosensor. ACS Synthetic Biology, 7(5), 1303–1314. https://doi.org/10.1021/ACSSYNBIO.7B00419/ASSET/IMAGES/MEDIUM/SB-2017-00419D\_M005.GIF
3. Homann S, Hofmann C, Gorin AM, et al. A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells. PLoS One. 2017;12(9). doi:10.1371/JOURNAL.PONE.0182941
4. Johansson, J., Mandin, P., Renzoni, A., Chiaruttini, C., Springer, M., & Cossart, P. (2002). An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell, 110(5), 551–561. https://doi.org/10.1016/S0092-8674(02)00905-4
5. Kortmann, J., Sczodrok, S., Rinnenthal, J., Schwalbe, H., & Narberhaus, F. (2011). Translation on demand by a simple RNA-based thermosensor. Nucleic Acids Research, 39(7), 2855. https://doi.org/10.1093/NAR/GKQ1252
6. Morita, M. T., Tanaka, Y., Kodama, T. S., Kyogoku, Y., Yanagi, H., & Yura, T. (1999). Translational induction of heat shock transcription factor σ32: evidence for a built-in RNA thermosensor. Genes & Development, 13(6), 655. https://doi.org/10.1101/GAD.13.6.655

### Plasmid extraction

*E. coli* DH5α or DH5α -Z1 cells harbouring the pLO\_SNAP, pET28, pSynSense2.5000 and pBAD plasmids were grown overnight in 5mL Luria-Bertani (LB) broth medium with 100 µg/mL ampicillin, 50 µg/mL kanamycin, 34 µg/mL and 50 µg/mL kanamycin respectively. The tubes were centrifuged for 10 minutes at 4000 g and 20°C, the supernatant was decanted. The plasmids are extracted from the cell pellets using the E.N.Z.A.® Plasmid DNA Mini Kit I (Omega Bio-tek, Georgia, USA) following the manufacturer’s instructions. The dsDNA concentration and quality were measured using the SPECTROstarNano (BGM Labtech, Ortenberg, Germany).

### Primer designs and polymerase chain reaction (PCR)

Primers were designed using SnapGene 5.08 ® software (from Insightful Science; available at snapgene.com) and checked for secondary structures and/or primer dimers using OligoAnalyzerTM Tool (IDT, Iowa, USA). The annealing sequence is 15-25 bp long and the melting temperature (Tm) of corresponding forward and reverse primers differed no more than 3°C. Overhangs are added to each primer depending on the intended cloning strategy. For type IIS assembly, the recognition site for BsaI-HF®-v2 was added. For Gibson assembly, the overhangs of the primer used to amplify the insert are homologous to the overhang of the primer used to amplify the vector. Primers were ordered from Integrated DNA Technologies (IDT, Iowa, USA). An overview of the PCR reaction mixtures, the thermocycler conditions and the extension time calculation can be found in tables 1, 2 and 3. 5X MyTaq reaction was obtained from Meridian Bioscience (Ohio, USA). Q5 reaction buffer, Q5® High-Fidelity polymerase, Taq polymerase and Q5 High GC enhancer were obtained from NEB (New England Biolabs, Massachusetts, USA). The Doppio thermocycler from VWR (Pennsylvania, USA) was used to perform the reactions.

Table 1: Primers designed for constructing the plasmids of the Eat-Shoot parts. gBlocks were ordered from IDT and TwistBioscience.

| Plasmid | Template | Primers Type IIS assembly |
| --- | --- | --- |
| pLO\_NarX mutant mPapaya pLO\_SNAP | 029\_FW\_pLO\_SHOOT3:  5’–AAAGCTCTTCATCTTACTGCGTCACGGATCTCCA | |
| | 061\_RV\_pLO\_Shoot Correct:  5’–AAAGCTCTTCAGAAGTGTATATCTCCTTTCTAG | |
| NarX mutant mPapaya gBlock | 035\_FW\_RBS5000\_NarX\_NarXmut:  5’–AAAGCTCTTCAATCGCTCACTCTTAAACTTCACTCGGG | |
| | 036\_RV\_NarXmut\_mPap:  5’–AAAGCTCTTCAGATCATAGCAAGCAGCTACAGC | |
| pSynsense2.5000\_NarX mNeonGreen\_PNarL mKate NarX mNeonGreen gBlock | 035\_FW\_RBS5000\_NarX\_NarXmut:  5’–AAAGCTCTTCAATCGCTCACTCTTAAACTTCACTCGGG | |
| | 036\_RV\_NarXmut\_mPap:  5’–AAAGCTCTTCAGATCATAGCAAGCAGCTACAGC | |
| pSynsense\_PNarL mKate | 033\_FW\_pNarL\_mKate:  5’–AAAGCTCTTCAATCAAGTCCAAGCGTTACATCTC | |
| | 038\_RV\_pSynSense:  5’–AAAGCTCTTCAGATCCGAGTGACGCTTAGGAGTGAGC | |
| pSynsense2.5000 NarX mutant mPapaya\_PNarL mKate  NarX mutant mPapaya gBlock | 035\_FW\_RBS5000\_NarX\_NarXmut:  5’–AAAGCTCTTCAATCGCTCACTCTTAAACTTCACTCGGG | |
| | 036\_RV\_NarXmut\_mPap:  5’–AAAGCTCTTCAGATCATAGCAAGCAGCTACAGC | |
| pSynsense\_PNarL mKate | 033\_FW\_pNarL\_mKate:  5’–AAAGCTCTTCAATCAAGTCCAAGCGTTACATCTC | |
| | 038\_RV\_pSynSense:  5’–AAAGCTCTTCAGATCCGAGTGACGCTTAGGAGTGAGC | |
| pSynsense\_PNarL mKate  pSynSense2.5000 | 037\_FW\_pSynSense:  5’–AAAGCTCTTCCTCTGCGGTTTGAAGGGTATTGG | |
| | 038\_RV\_pSynSense:  5’–AAAGCTCTTCAGATCCGAGTGACGCTTAGGAGTGAGC | |
| PNarL mKate | 033\_FW\_pNarL\_mKate:  5’–AAAGCTCTTCAATCAAGTCCAAGCGTTACATCTC | |
| | 034\_RV\_pNarL\_mKate:  5’–AAAGCTCTTCAAGACATAGCAAGCAGCTACAGC | |

Table 2: Primers designed for constructing the plasmids of the Leave part. gBlocks were ordered from IDT and TwistBioscience.

| Intended construct | Template | Primers Type IIS assembly |
| --- | --- | --- |
| pLO\_Toxin, pLO\_Hsp17, pLO\_rpoH, pLO\_prfA pLO\_SNAP | 008\_FW\_pLO:  5’-AAAGGTCTCGGCACCTACTGCGTCACGGATCTCC | |
| | 017\_RV\_pLO insert:  5’–AAAGGTCTCGACTGGGGTCATGTGAGCAA | |
| Toxin gblock, hsp17 gblock, rpoH gblock, prfa\_CcdB\_sfGFP | 018\_FW\_T/TA\_ECOLI:  5’–AAAGGTCTCCCAGTGTATGCGATCTACTGCTACTTCG | |
| | 002\_RV\_T/TA\_ECOLI:  5’–AAAGGTCTCAGTGCCAGCACTACGCTACGGACTA | |
| pLO\_Toxin\_LacI pLO\_Toxin | 021\_FW\_pLO\_LacI:  5’–AAAGGTCTCGTGACTTCGTTCCACTGAGCGTCAGAC | |
| | 006\_RV\_pLO\_LacI:  5’–AAAGGTCTCGGCACCACGTTAAGGGATTTTGGTCATG | |
| pET28 | 013\_FW\_LAQI:  5’–AAAGGTCTCGGTCACCTAATGAGTGAG | |
| | 014\_RV\_LAQI:  5’–AAAGGTCTCAGTGCTGGCGGAGCTGAATT | |
| prfA\_CcdB\_sfGFP prfA gBlock | 018\_FW\_T/TA\_ECOLI:  5’–AAAGGTCTCCCAGTGTATGCGATCTACTGCTACTTCG | |
| | 019\_RV\_prfA:  5’–AAAGGTCTCATGACTGCTTGAGCGTTCATGTCT | |
| hsp17 gBlock | 020\_FW\_ccdB\_GFP:  5’–AAAGGTCTCAGTCAATGAAGCAACGCATTACTGT | |
| | 002\_RV\_T/TA\_ECOLI:  5’–AAAGGTCTCAGTGCCAGCACTACGCTACGGACTA | |
| pBAD\_Toxin pBAD | 050\_FW\_pBAD\_insert:  5’–AAAGGTCTCGGCACACTCAGAAGTGAAACGTCGTAGC | |
| | 049\_RV\_pBAD\_insert:  5’–AAAGGTCTCGACTGCCCCGCAGATGTATATCTCCTTC | |
| Toxin gBlock | 018\_FW\_T/TA\_ECOLI:  5’–AAAGGTCTCCCAGTGTATGCGATCTACTGCTACTTCG | |
| | 002\_RV\_T/TA\_ECOLI:  5’–AAAGGTCTCAGTGCCAGCACTACGCTACGGACTA | |

Table 3: PCR protocols: reaction mixture (50 µL)

| Q5® HF PCR | Q5® HF PCR + formamide | Q5® High-Fidelity 2X Master Mix | Taq PCR |
| --- | --- | --- | --- |
| 10 µL 5x Q5 reaction buffer 10 µL 5x Q5 reaction buffer | 25 µL Q5 High-Fidelity 2X Master Mix | 5 µL 10X Standard Taq Reaction Buffer | |
| 1 µL 10 mM dNTPs 1 µL 10 mM dNTPs |  | 1 µL 10 mM dNTPs | |
| 2 µL 10 µM Fp 2 µL 10 µM Fp | 2.5 µL 10 µM Fp | 1µL 10 µM Fp | |
| 2 µL 10 µM Rp 2 µL 10 µM Rp | 2.5 µL 10 µM Rp | 1µL 10 µM Rp | |
| 5 µL 1 ng/µL template DNA 5 µL 1 ng/µL template DNA | 5 µL 1 ng/µL template DNA | 5 µL 1 ng/µL template DNA | |
| 1 µL Q5 HF polymerase 1 µL Q5 HF polymerase |  | 0.25 µL Taq DNA polymerase | |
| 10 µL Q5 High GC enhancer 10 µL Q5 High GC enhancer |  |  | |
| 19 µL mQ water 18.5 µL mQ water | 15 µL mQ water | 36.75 µL mQ water | |
| 0.5 µL formamide |  |  | |

Table 4: PCR protocols: thermocycler conditions

|  | Q5® HF PCR | Taq PCR |
| --- | --- | --- |
| Initial denaturation 98°C, 30s - Colony PCR: 5 minutes | 95°C, 30s - Colony PCR: 5 minutes | |
| *START CYCLE -*  Number of cycles 32 | 30 | |
| Denaturation 98°C, 10s | 95°C, 20s | |
| Annealing Tm, 20s | Tm, 20s | |
| Extension 72°C, calculate extension time | 68°C, calculate extension time | |
| *END CYCLE*  |  | |
| Final extension 72°C, 5 min | 68°C, 5 min | |
| Storage 16°C, infinite | 16°C, infinite | |

Table 5: Calculation of the extension time: Q5 HF polymerase PCR reaction

| Length | Time |
| --- | --- |
| ≤ 1 Kb 15 seconds | |
| (1 Kb; 5 Kb) 30 seconds/Kb + 30 seconds | |
| (5 Kb; 9 kb) 1 minute/Kb | |
| (9 Kb; 10 kb) 1.5 minute/Kb | |

Table 6: Calculation of the extension time: Taq polymerase PCR reaction

| All fragments | 1 minute/Kb |
| --- | --- |

To analyze whether the desired fragment length was amplified, an agarose gel electrophoresis is carried out. Depending on the expected length, a 1.0% (400 bp – 10kb) or a 2.0% (100 bp – 2 kb) agarose gel is prepared according to the manufacturer’s instructions. 0.50 g (1.0% gel) or 1.00 g (2.0% gel) UltraPure™ Agarose (Invitrogen) was added to 50 mL LiAc (Lithium acetate) buffer and microwaved for 1-2 minutes. 4µL SYBR Safe DNA Gel Stain (Invitrogen) was added to the cleared agarose solution which was casted and left for 10 minutes to polymerize. The 1 kb DNA ladder (all obtained from NEB) was always used as a reference. Samples were prepared by adding 2 µL of 6X Gel Loading Dye Purple (NEB) to 10 µL of the PCR reaction mixture.

### DNA extraction

PCR products were extracted from the PCR reaction mix with the MicroElute® Cycle – Pure Kit (Omega Bio-tek, Georgia, USA) according to the manufacturer’s instructions. When multiple fragments are visualised using agarose gel electrophoresis, the Monarch® DNA Gel Extraction Kit (New England Biolabs, Massachusetts, USA) was used to extract the desired fragments.

### Type IIS cloning The following digestion mix (50µL) was set up on ice: 1µg DNA, 5µL 10X NEB buffer r3.1, 1µL type IIS restriction enzyme, mQ water up to 50µL. The reaction was incubated at the at 37°C for 1 hour followed by a heat inactivation at 65°C for 20 minutes. All enzymes and reaction buffers were obtained from NEB. After digestion, DNA was extracted using the appropriate technique (NEBcloner, 2022). Alternatively, heat inactivation can be skipped to protect the sample when purification and ligation are done right after digestion. It is also possible to premix the digested products before purification. The enzymes used were BsaI-HF, HindIII-HF, NcoI-HF and SalI-HF. BsaI-HF was used during the cloning process, SalI-HF was used to remove the stopcodon from the toxin construct and finally HindIII-HF and NcoI-HF were used to linearise plasmids in order to visualize them with agarose gel electrophoresis. The following ligation reaction (20µL) was set up on ice: 1µL 10X T4 DNA ligase buffer, 50 ng vector DNA (4 kb), 37.5 ng insert DNA (1 kb), 1µL T4 DNA ligase, mQ water up to 20µL. The vector: insert ratios were optimized according to the lengths with the NEBioCalculator. For troubleshooting, different ratios of insert and vector can be tried. The reaction was incubated at room temperature for 10 minutes and the enzyme was inactivated at 65°C for 20 minutes (NEBcloner, 2022). Before transformation, the sample is stored at -20°C. All enzymes and reaction buffers were obtained from NEB. Gibson assembly To perform Gibson assembly, the following reaction mixture (20µL) was set up: 0.02 – 0.5 pmols of DNA (total amount of fragments), 10 µL 2X Gibson Assembly Master Mix, mQ water up to 20 µL. To optimize the cloning efficiency, 50-100 ng of vector DNA was added to a 2-3 fold excess of insert DNA. The sample was incubated at 50°C for 15 minutes and stored at -20°C before transformation (Gibson Assembly® | NEB, 2022). Transformation 50 µL of chemically competent cells (stored at -80°C) were thawed on ice and 1 pg – 100 ng of plasmid DNA was added. The tube was then flicked 4-5 times and the mixture was put on ice for 5 to 30 minutes. Following this, the sample was heat shocked at 42°C for 30 to 60 seconds and the sample was put back on ice for 5 minutes. Then, 450 µL of sterile LB medium was added to the cell suspension and this was incubated at 37°C for 1 hour while shaking. Lastly, 25 to 100 µL of this sample was plated on the appropriate selection plate which was then incubated at 37°C overnight. DH5α cells were obtained from NEB or competent cells were prepared using the following protocol. Alternatively, when difficulties occur, the sample can be incubated without shaking and avoid pipetting up and down to mix. After plating 25 to 100 µL, centrifuge the remaining cells and discard the supernatant. Resuspend with 100 µL LB medium and plate this on another plate. Competent cell preparation 500 µL of an overnight cell culture (3mL LB with kanamycin, 37°C, 180 rpm) was diluted in a sterile Erlenmeyer containing 50 mL LB. It was incubated at 37°C until the cells were grown to an OD600 of 0.2 – 0.6. The cells were cooled down on ice and centrifuged (4°C, 5000 rpm, 10 min). The cell pellet was then resuspended in ice cold, sterile 0.1 M CaCl2 and put on ice for at least 25 min. Following this, the cells were centrifuged again (4°C, 5000 rpm, 10 min) and the pellet resuspended in 500 µL ice cold, sterile 0.1 M CaCl2. 100 µL ice cold, sterile 80% glycerol was added to this solution. Lastly, this cell suspension was divided into sterile Eppendorf tubes (50 µL each) and stored at -80°C. Colony PCR and sequencing After transformation, colonies were picked with a swab which was washed in the PCR reaction mixture. Under sterile conditions, it is streaked onto a new LB agar selection plate which is incubated at 37°C overnight. The PCR tubes were incubated for 2 minutes at 95°C to lyse the cells which was followed by the appropriate thermocycling conditions. Two primers were designed to amplify a region where the insert and the vector are ligated. The correct length can be verified with an agarose gel and if the amplicon had the expected length, the samples were sequenced. The sample preparation for Sanger sequencing was done according to the instructions of Eurofins. Induction tests *TOXIN CONSTRUCTS -* To test the toxin constructs, *E. coli* DH5αZ1 cells containing different plasmids are grown overnight (37°C, LB medium). The overnight cultures were diluted 50X in 96 well plates (TPP Tissue culture test plate 96F, Techno Plastic Products AP) and grown until they reached an OD600 of 0.2 - 0.6. OD600 and fluorescence measurements were done with the CLARIOstar (BMG Labtech, Ortenberg, Germany). All measurements were performed in triplicate. Four colonies were tested either with the plasmid pLO\_toxin or pBAD\_toxin. An overview of the different test conditions is provided in the table below. Table 5: Different test conditions for the toxin constructs. | Test condition | Medium composition | | --- | --- | | Blanks LB with the appropriate antibiotic with the different concentrations of inducers | | | Negative control *E. coli* DH5α-Z1, no plasmid, LB | | | Test media: LB with appropriate antibiotic No inducer | | | IPTG 0.1 mM | | | IPTG 0.5 mM | | | IPTG 1 mM | | | Anhydrotetracycline (aTC) 50 ng/mL | | | aTC 100 ng/mL | | | aTC 200 ng/mL | | | IPTG 0.1 mM + aTc 50 ng/mL | | | IPTG 0.5 mM + aTc 100 ng/mL | | | IPTG 1 mM + aTc 200 ng/mL | | *ANTITOXIN CONSTRUCTS* - Two 96 well plates were prepared with 200 µL of the relevant selection medium (LB + 100 µg/mL ampicillin) and the plates were covered with a breathable film. The cultures were diluted 50 times in these well plates and incubated at different temperatures overnight at 180 rpm. Temperatures tested ranged from 20°C to 42°C. The next day, both the OD600 as the fluorescence intensity were measured with the CLARIOstar (BMG Labtech, Ortenberg, Germany). Blanks (LB) and negative controls (culture with pLO\_SNAP plasmid) are subjected to the same protocol. All measurements were performed in triplicate. References 1. Gibson Assembly® | NEB. (2022). New England Biolabs. Geraadpleegd op 9 augustus 2022, van https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly 2. NEBcloner. (2024). New England Biolabs. Geraadpleegd op 29 juni 2022, van https://nebcloner.neb.com/#!/ 3. NEBioCalculator. (z.d.). New England Biolabs. Geraadpleegd op 6 augustus 2022, van https://nebiocalculator.neb.com/#!/ligation

# Overview

This part explains the lab results of the different subprojects. More information regarding the conditions and interpretation of the experiments can be found under Notebook and Engineering success.

### Shoot Results

In this part of the project, we are utilizing pSynSense2.5000 developed by Prof. De Mey’s research group at UGent. Following their suggestion, we sequenced the full plasmid and aligned it to the map they provided in the literature (De Paepe et al., 2018 [[1]](#reference)). The sequencing resultshowed that it aligns with the map. The PCR of pSynSense2.5000 was done afterwards. As the complementary plasmid, pLO\_SNAP was also amplified as the backbone of the NarX mutant construct. Both plasmid amplification showed the desired length of 3223 bp for pLO and 5478 bp for pSynSense2.5000 (Figure 1).

![](https://static.igem.wiki/teams/4345/wiki/results/resultsshoot1.png)

Figure 1: Amplification of pLO and pSynSense2.5000 backbones

gBlocks of PNarL-mKATE, NarX-mNeonGreen, and NarX mutant-mPapaya were also amplified. All of the PCR showed the desired product length of 885 bp and 2733 bp respectively (Figure 2).

All of the PCR products were isolated, digested using SapI, and ligated using T4 ligase. Then the constructs (pLO\_NarX mutant-mPapaya, pSynSense2.5000\_NarX-mNeonGreen\_PNarL-mKATE, and pSynSense2.5000\_NarX mutant-mPapaya\_PNarL-mKATE) were transformed into *E. coli* DH5α. Colonies were obtained on the selection plates afterwards except for pLO\_NarX mutant mPapaya (Figure 3).

![](https://static.igem.wiki/teams/4345/wiki/results/resultsshoot2.png)

Figure 2: Amplification of gBlocks

![](https://static.igem.wiki/teams/4345/wiki/results/shoot3.png)

Figure 3: pSynSense2.5000 with NarX and NarX mutant inserts

Colony PCR were done afterwards for the colonies obtained. The primers anneal on the plasmid region that flank the insert thus the desired length is 4.1 Kb. However, the result showed bands lower than 1.5 Kb (Figure 4).

![](https://static.igem.wiki/teams/4345/wiki/results/shoot4.png)

Figure 4: Colony PCR result of pSynSense2.5000

After sent for sequencing, the plasmid showed that only PNarL-mKATE is inserted (Figure 5). NarX-mNeonGreen and NarX-mPapaya were not included in the construct. This was due to the same sequences use for the type IIS overhangs. However, the SapI cut sites were not present anymore in the construct because of the type IIS restriction enzyme feature. Thus, the newly assembled pSynSense2.5000\_PNarL-mKATE were amplified, digested, and ligated with the insert. Construction of pLO\_NarX mutant-mPapayawas also done in parallel.

![](https://static.igem.wiki/teams/4345/wiki/results/sequencing.png)

Figure 5: Sequencing result of pSynSense\_PNarL-mKATE

Colonies of pLO\_ NarX mutant-mPapaya were obtained but didn’t show a positive result in the colony PCR. Meanwhile, pSynSense2.5000 constructs were harder to clone. However, colonies were obtained in the last transformation. However, due to the time constrains, validation using colony PCR and sequencing were not yet done. Therefore, the construct assembly of Shoot is still incomplete.

References

1. De Paepe, B., Maertens, J., Vanholme, B., & de Mey, M. (2018). Modularization and Response Curve Engineering of a Naringenin-Responsive Transcriptional Biosensor. ACS Synthetic Biology, 7(5), 1303–1314.

### Leave Results

### Toxin

### Cloning of Toxin into pLO\_SNAP

PCR of the Toxin gblock and pLO\_SNAP plasmid is done to introduce the BsaI restriction sites using overhangs in the primers. Please look at the results of the antitoxin for the gel image of the Toxin and pLO\_SNAP PCR productsbecause the experiment was done in parallel. Digestion is done with the restriction enzyme BsaI for type IIS cloning. Hereafter, the insert and vector were ligated, respectively. The transformation was done in the *E. coli* strain DH5α. The plasmid of the positive colonies is purified and also transformed into the *E. coli* strain DH5α.
A problem occurred with colony PCR (primers 9 and 10). After it failed repeatedly, there was decided to send them for sequencing (Eurofins) because they kept growing on an antibiotic selective plate. Sequencing results turned out to be successful for multiple colonies. Two were chosen to go further with the testing. The plasmid of the positive colonies is purified and also transformed into the *E. coli* strain DH5α.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin1.png)

Figure 6: Visualization of sequencing results of pLO\_Toxin insert, generated with Snapgene

### Cloning of LacI into pLO\_Toxin

The already successfully cloned pLO\_Toxin plasmid is amplified by PCR to introduce BsaI restriction sites for Type IIS cloning. Besides this, the LacI gene is amplified from the pET28 plasmid for the same reason. The restriction sites and compatible overhangs are introduced using primers with specific overhangs (see Engineering). Because of the unexpected bands on the Agarose gel of the PCR of pET28, gel extraction was done with the band of interest (1025bp). After a PCR with the extracts, the gel showed a single clear band as can be seen in figure 9.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin2.png)

Figure 7: Agarose gel of PCR product pLO\_Toxin with BsaI restriction sites Template: pLO\_Toxin
Primers: 021 and 006; 4110 bp

![](https://static.igem.wiki/teams/4345/wiki/results/toxin3.png)

Figure 8: Agarose gel of LacI gene PCR out of pET28 plasmid with BsaI restriction sites
primers 013 and 014

PCR products are purified and after digestion with BsaI, ligation of the insert LacI and vector pLO\_Toxin was carried out. Hereafter, the ligation product was transformed into DH5α competent cells and incubated overnight. Colony PCR showed all positive hits as can be seen in figure 10.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin4.png)

Figure 9: Agarose gel of the LacI gene PCR products out of gel purification, with BsaI restriction sites
Primers: 13 and 14; 1025 bp

![](https://static.igem.wiki/teams/4345/wiki/results/toxin5.png)

Figure 10: Agarose gel of Colony PCR pLO\_Toxin\_LacI

The plasmids of selected colonies are purified and sent for sequencing (Eurofins). Ligation of the second colony seemed to besuccessful but showed a single point mutation in the startcodon of the LacI gene as shown in figure 11, derived from Snapgene. Because of this, the toxin design took another approach by using the *E. coli* DH5αZ1 strain that contains the LacI gene in its genome, as explained in Engineering Success.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin6.png)

Figure 11: Sequencing results of pLO\_LacI

### Cloning of Toxin into pBAD

Another plasmid called pBAD was used to insert the Toxin gene. The reasoning behind this can be found in Engineering Success.
PCR of the plasmid was carried out to introduce BsaIrestriction sites with primers containing overhangs.
The PCR products are digested with BsaI, purified and ligated. This is followed by transformation into an *E. coli* DH5αZ1 strain. Because of time constraints, no successful Colony PCR or sequencing was done. It was decided to proceed with testingthe construct for fluorescence after induction.
Because of time constraints, it was not possible to do a successful colony PCR of pBAD\_Toxin. Because of this, it was not possible to sequence the construct. It was decided to proceed with testing the construct for fluorescence after induction.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin7.png)

Figure 12: Agarose gel of pBAD PCR products with BsaI restriction sites
Primers: 049 and 050; 3685 bp

![](https://static.igem.wiki/teams/4345/wiki/results/toxin8.png)

Figure 13: Agarose gel of gblock Toxin PCR product with BsaI restriction sites
Primers: 002 and 018; 1437 bp

### Cloning of Toxin into pLO\_SNAP

PCR of the Toxin gblock and pLO\_SNAP plasmid is done to introduce the BsaI restrictionsites using overhangs in the primers. Please look at the results of the antitoxin for the gel image of the Toxin PCR products because the experiment was done in parallel. Digestion is done with the restriction enzyme BsaI for type IIS cloning. Hereafter, the insert and vector were ligated, respectively. The transformation was done in the *E. coli* strain DH5α. The plasmid of the positive colonies is purified and also transformed into the *E. coli* strain DH5α.
A problem occurred with colony PCR (primers 009 and 010). After three tries there was decided to send them for sequencing (Eurofins) because they kept growing on an antibiotic selective plate. Sequencing results turned out to be successful for multiple colonies. Two were chosen to go further with the testing. The plasmid of the positive colonies is purified and also transformed into the *E. coli* strain DH5α.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin9.png)

Figure 14: Visualization of successful sequencing results for pLO\_Toxin insert, generated with Snapgene

### Stop codon control deletion

To measure fluorescence, the construct has to be transcribed. In order to make this possible, the stop codon control region has to be deleted. This is done by restriction digestion with SalI followed by religation. Because of time constraints, the stop codon region was only deleted from the pLO\_Toxin but not from pBAD\_Toxin, which led to contradictory fluorescent results.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin10.png)

Figure 15: Visualization of successful sequencing results for the deletion of the stop codon control, generated by Snapgene

### Fluorescent measurement

After confirmed by sequencing, the pLO\_Toxin purified plasmid was isolated and cloned into an *E. coli* DH5αZ1 strain. Fluorescense measurement after induction with anhydrotetracyclin and IPTG is compared with pBAD\_Toxin, a plasmid that is also transformed into the *E. coli* DH5αZ1 strain. The insert of these two plasmids are the same toxin CcdB fused to sfGFP. However, pBAD\_Toxin still contains the stop codons.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin11.png)

Figure 16: Single induction fluorescence of pLO\_Toxin and pBAD\_Toxin

The toxin in both constructs is controlled by TetR, LacI, and stop codons. As mentioned in the experiments, different types and concentrations of inducer are tested. The data shown in Figure 16 is analysed on PRISM using paired t-test. There is a significant difference between the samples with the negative control indicating the presence of fluorescence. The fluorescence increases in parallel to the increasing concentration of anhydrous tetracycline (aTc). However, there are no significant difference between the increasing concentrations of IPTG.

![](https://static.igem.wiki/teams/4345/wiki/results/toxin12.png)

Figure 17: Combination induction fluorescence of pLO\_Toxin and pBAD\_Toxin

When induced using two inducers, pLO\_Toxin samples don’t show any fluorescence. While pBAD\_Toxin shows a significant and high fluorescence compared to the control. However, there are no significant differences between the treatments. These findings are visualized in figure 17. According to the result, we suspect that the toxin in pLO is expressed hence killing the cell which cause the absence of fluorescence. This explains why the fluorescence is still present when one inducer is being used. Meanwhile, pBAD\_Toxin depicts how high the toxin is expressed with the combinatorial induction, of IPTG and aTc, compared to single inducer in the previous graph. Raw data of these measurements are accesible[here.](https://static.igem.wiki/teams/4345/wiki/toxin-raw-data.pdf)

### AntiToxin

### Insert hsp17 and rpoH into pLO\_SNAP

PCR of pLO\_SNAP and the gblocks of the antitoxins is carried out to introduce the BsaI restrictionsites for type IIS cloningusing primer overhangs. Formamide was added to enhance PCR in some of the samples. Agarose gels can be found in figure 18 and 19.

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin1.png)

Figure 18: Agarose gel of PCR product gblocks of various constructs withBsaI restriction sites
Primers: 002 and 018
hsp17: 1327 bp
rpoH: 1529 bp
Toxin: 1437 bp

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin2.png)

Figure 19: Agarose gel of PCR products pLO\_SNAP with BsaI restriction sites
Primers: 008 and 017; 2934 bp

Restriction digestion for type IIS cloning was done with all PCR products to make compatible overhangs for sticky end ligation. After ligation, the constructs are transformed into the *E. coli* DH5α strain and incubated overnight. Colony PCR was carried out, positive colonies were selected, the plasmid was purified and sentfor sequencing.

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin3.png)

Figure 20: Visualization of successful sequencing results for pLO\_rpoH, generated with Snapgene

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin4.png)

Figure 21: Visualization of successful sequencing results for pLO\_hsp17, generated with Snapgene

### Insert prfA into pLO\_SNAP

It was not possible to order the gblock of the prfA RNA thermometer due to the formation of secondary structures. A gblock of the RNA thermometer prfA linked to CcdB was ordered and the promotor, RBS and prfA RNA thermometer sequence was amplified as one part using PCR with primers 018 and 019. CcdB linked to sfGFP was taken out of the hsp17 gblock (hsp17\_CcdA-sfGFP) by PCR to construct pLO\_prfA.

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin5.png)

Figure 22: Agarose gel of all PCR products needed for prfA insertion into pLO\_SNAP
primers pLO\_SNAP: 008 and 0017; 2934 bp
primers prfA: 018 and 019; 291 bp
primers hsp17\_CcdA-sfGFP: 002 and 020; 1144 bp

Restriction sites for BsaI are introduced with PCR using primers with specific overhangs. The part containing the specific RNA thermometer is ligated to the antitoxin part taken out of hsp17. This part is then ligated into the pLO\_SNAP plasmid and transformed into *E. coli* DH5α.

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin6.png)

Figure 23: Agarose gel of purified ligation product prfA\_hsp17\_CcdA-sfGFP

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin7.png)

Figure 24: Agarose gel of Colony PCR pLO\_prfA

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin8.png)

Figure 25: Visualization of successful sequencing results for pLO\_prfA, generated with Snapgene

### Fluorescent measurements

Various anti-toxin thermometers were tested via ClarioStar (BMG LABTECH). Testing was performed in triplicate per measurement. Negative controls were added to measure autofluorescence of the cells using an empty pLO vector in DH5α. Optical density was measured for both samples and negative controls, then results were normalized by OD. Our results for the original constructs can be seen in figure 26. Included are negative controls, which represent autofluorescence to compare the results. Because each temperature was only tested one to two times, only general statements can be made on the behaviours of our riboswitch thermometers. Values are normalized by OD, which were between the 0.2 and 0.8 range. Well plates were used and incubated at varying temperatures for 16 hours. Additionally, we filtered out results that were anomalous compared to others within the same temperature. Looking at figure 26 we can see that there is a general trend of greater fluorescence as temperature increase, most notably at 42°C compared to the autofluorescence.

![](https://static.igem.wiki/teams/4345/wiki/engineering/leave/figure-28.png)

Figure 26: Fluorescence by original construct and temperature.

Images of plates were taken along-side each measurement, shown in Figures 27-28. Across all constructs, visually only hsp17 col2 showed any fluorescence to the naked eye.

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin10.jpg)

Figure 27a

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin11.jpg)

Figure 27b

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin12.jpg)

Figure 28a

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin13.png)

Figure 28b

Hsp17 col2 was the most promising thermometer. As such, it was tested in additional strains MG1665 and BL22. The plasmid was extracted and purified then transformed into MG1665 and BL22. Colony PCR was done to determine if transformation was successful. An image for the plate for the colony PCR is shown in figure 29

![](https://static.igem.wiki/teams/4345/wiki/results/antitoxin14.png)

Figure 29

![](https://static.igem.wiki/teams/4345/wiki/engineering/leave/figure-29.png)

Figure 30: Hsp17 col2 plasmids in two new strains

The results of these temperature tests can be seen in Figure 30. Both hsp17 col2 plasmid in MG1665 and BL22 rapidly increase in fluorescence between 31°C and 37°C. Compared to the autofluorescence of the negative control, the values between 31°C and 37°C for the strains MG1665 and BL22 are more than triple.

Images of plates were taken along-side each measurement, shown in Figure 31. Across all constructs, visually only hsp17 col2 showed any fluorescence to the naked eye.

![](https://static.igem.wiki/teams/4345/wiki/engineering/leave/figure-30.jpg)

Figure 31: Plate images of hsp17 different temperatures

To download the data for the results of AT thermometer temperature tests, access [**Here**](https://doi.org/10.5281/zenodo.7186933)

[![DOI](https://zenodo.org/badge/DOI/10.5281/zenodo.7186933.svg)](https://doi.org/10.5281/zenodo.7186933)

### Conclusions

In conclusion, the Leave part of our project showed the possibility of utilizing a kill switch based on temperature as a biocontainment method. From the three riboswitches we tested, hsp17 is the most promising RNA thermometer. However, due to some inconsistent data and limited repetition, more testing is needed to completely characterize the different kill switches used.

As for the toxin part, the induction data looks promising. However, the measurements were only done once and need to be repeated several times to ensure the validity of the result. Furthermore, co-transformation with the antitoxin plasmid is needed to confirm the outcome of our designs combined.

Finally for the Shoot part, we were not able to finalize the construction of our plasmid due to the lack of time. However, if the transformation is successful, microscopy and measurements of mKATE expression will be interesting to investigate further.

References

1. Lee, T. S., Krupa, R. A., Zhang, F., Hajimorad, M., Holtz, W. J., Prasad, N., Lee, S. K., & Keasling, J. D. (2011). BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Journal of biological engineering, 5, 12. https://doi.org/10.1186/1754-1611-5-12
2. Hannon, G. (2002). RNA interference. Nature 418, 244–251. https://doi.org/10.1038/418244a
3. RNA fold Webserver. http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3&ID=MLjhUGXlaY
4. O-acetyl-ADP-ribose deacetylase (YmdB). Uniprot. https://www.uniprot.org/uniprot/P0A8D6