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

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).

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 accesiblehere.

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.

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.

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.

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.

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.

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.

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

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.

To download the data for the results of AT thermometer temperature tests, access Here

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.