Part:BBa_K4868001

PaqCI cloning site with Fluoride detection insertion sequence

The part consists of a T7 promoter (BBa_J64997), lac operator(BBa_J64997), RBS(BBa_K2621038), an insertion sequence (RFP (BBa_K4868002)) followed by a His-tag (BBa_J18909) and a T7 terminator (BBa_K731721), an Anderson promoter (BBa_J23119), fluoride-dependent riboswitch(K4868000), super-folder green fluorescent protein (sfGFP) (BBa_K2541400), a T1 terminator (BBa_B0010) and a T7 terminator (K731721). The insertion sequence allows for the insertion of a given gene using Golden Gate cloning. The sequence, which is removed in this process encodes an RFP, and can therefore be used in selecting successful cloning. The riboswitch allows translation of the downstream sfGFP expression in the presence of fluoride ions. The use of this composite part is, therefore, to insert an enzyme that influences fluoride concentration, and then be able to use concentration-dependent fluorescence to select.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 941
  Illegal NheI site found at 964
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1786
  Illegal XhoI site found at 1486
  Illegal XhoI site found at 1795
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 750
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage

A recent interest in polyfluorinated compounds, PFAS, has risen. They are notoriously difficult to degrade enzymatically, and it is therefore of great interest for iGEM teams to have methods of evaluating the degree of degradation. A major component of degradation is the cleavage of the C-F bonds in such compounds. This part works on the basis of the release of fluoride and gives a means of quantifying the release of fluoride, which is why we will use the part as expression plasmid of our Dehalogenases; DeHa1 (BBa_K4868996), DeHa2 (BBa_K4868997), DeHa (BBa_K4868998), and DeHa5 (BBa_K4868999).

This became a central part of our project - being able to select cells based on fluoride release from enzymatic degradation of perfluorooctanoic acid (PFOA). Our team worked with the optimization of fluoroacetate dehalogenases, and our design allows the release of fluoride from the enzyme activity to result in increased fluorescence by activation of the riboswitch. The sfGFP came to be a major component in our experimental setup, as it allowed for separation/selection of cells based on their fluorescence with FACS, on a quantifiable level. The insertion site allows for modular insertion of enzymes, but also various methods of evaluating the success of insertion. The RFP can be detected in colonies, indicating unsuccessful insertion at a quick glance, while it is still possible to detect the fragment with gel electrophoresis if the fragment has a different size than RFP or the primers used to multiply the fragment bind within the fragment sequence.

Future iGEM teams working with degradation of PFAS can readily use this part to easily evaluate the release of fluoride with fluorescence microscopy, FACS or fluorescence measurement by a plate reader. The insertion site allows for future iGEM teams to work with any enzyme they wish, in a context involving changes in fluoride concentrations, such as fluoride-releasing reaction, transport of fluoride across the cell membrane, and biosensors.

Biology

The riboswitch used in this part is a fluoride-sensitive riboswitch from B. cereus shown to function with E. coli RNA polymerase transcription(Thavarajah et al., 2020). In this species, it functions in regulating the expression of the crcB gene, which functions as a fluoride efflux pump. Fluoride induces a structural change in the transcribed riboswitch, allowing for continued transcription. The secondary structure formed by the binding of fluoride to the aptamer region induces a conformational change into an anti-terminating pseudoknot. The absence of fluoride leaves the transcribed riboswitch in a terminating secondary structure, stopping the elongation of the transcript(Lee et al., 2021). (Figure 1)

Charaterization

Assembly of the part

The first step to using this composite part is assembling it into a backbone. This part had to be ordered in two parts since it could not be synthesized in one piece. Therefore, Gibson assembly was the first step in the assembly of the part. For the Gibson assembly, the pET-51b(+) backbone had to be linearized first. For this the restriction enzymes SgrAI (NEB #R0603S) and HindIII (NEB #R3104) were used, to cut out the no longer needed part of the plasmid, and the linearized backbone was purified from a gel. The overlap regions on the part were designed according to the linearized plasmid after restriction digestion. The assembly itself was done by mixing vector and insert DNA to a ratio of 1:2 (vector : insert) that have the total amount of fragments being 0.03-0.2 pmol with 10 µL of the High-fidelity assembly master mix (NEB #E2621S). There was added deionized water to a total volume of 20 µL, after which the reaction was incubated at 50°C for 15 minutes. After incubation the mixture was ready to be transformed into competent cells. Since our part includes RFP controlled by a T7 promoter, the transformation was done into competent cells that have the T7 expression system in their genomic DNA (NEB #C2566I). With the assembled plasmid in a T7 expression strain, it was possible to see which colonies contained and expressed RFP under the induction of 0.4 mM IPTG. There was also 100 µg/mL ampicillin in the selection plates since that was the resistance gene in the pET-51b(+) backbone. Figure 2 shows the transformation plate exposed to blue light, which leads to the excitation of RFP, making it very easy to distinguish which colonies express RFP and were thereby assembled correctly.

Gibson assembly of BBa_K4868001 into pET-51b(+)

Figure 2: Fluorescence assessment of RFP under blue light exposure after Gibson assembly of BBa_K4868001 into pET-51b(+). The fluorescent colonies have most likely successfully inserted BBa_K4868001.

To further test the reliability of selecting correct assemblies by selecting fluorescent colonies, a colony PCR was made, where the appurtenant gel can be seen in figure 2. The primers for the PCR were designed to bind to the backbone on either side of the insertion piece and the amplified region has a length of 2004 bp.

Forward primer: 5’ AAATTGAGGAGAAGCCCGG

Reverse primer: 5’ ATATAGGCGCCAGCAACC

For the PCR, the Ampliqon 2x Taq red master mix (Cat. No.: A190303) was used and the following PCR program was run:

Step:	Temperature:	Time:	Cycles
Denaturation	95°C	2 min	--
Denaturation	95°C	25 sec	30x
Annealing	51°C	30 sec
Elongation	72°C	2 min
Elongation	72°C	5 min	--

The gel seen in figure 3 shows the PCR gel, where the wells 1-8 there are samples where fluorescent colonies were selected for the PCR. These show the same length as the bands in well 9&10, which are positive controls of plasmids that through DNA sequencing had confirmed that BBa_K4868001 was inserted correctly into the plasmid backbone. The wells 11&12 show the PCR product of the WT strain, which is the negative control, since it shows that no genomic DNA is being amplified.

Figure 3: PCR gel electrophoresis of Gibson assembly transformation. 1-8 are samples of fluorescent colonies, 9&10 confirmed correctly assembled plasmids with BBa_K4868001 (positive control), 11&12 are WT (negative control), 13 H2O and primer contamination control.

In well 13 there is a weak band, which indicates that either the water or primers are contaminated, but the band size is too large to be the insertion piece so it should not have affected the other samples. The fluorescence selection is primarily a method to be able to only pick colonies that have received the assembled plasmid. However, for certainty of correct assembly, and to be sure that no mutations have happened, it is important to sequence the insertion sequence.

Golden gate cloning using PaqCI

One of the key parts of BBa_K4868001 is the PaqCI (NEB #R0745S) insertion site. PaqCI is a type-II restriction enzyme, which is a class of restriction enzymes that have a very high cloning efficiency. Another advantage of type-II restriction enzymes is that the plasmids won’t be able to re-ligate itself without the matching insert because the recognition site isn’t the same as the restriction site and the restriction site can manually be modified. The overhangs created by the restriction sites have been carefully designed on both the insertion sequence and our enzymes (DeHa 1 (BBa_K4868996), DeHa 2 (BBa_K4868997), DeHa 4(BBa_K4868998), and DeHa 5 (BBa_K4868999)) that we are inserting into the plasmid, guaranteeing that the only two possible outcomes after golden gate cloning are colonies that have received uncut plasmids and colonies that have received correctly assembled plasmid. Because of the RFP that is in the PaqCI insertion site, it will be easy to distinguish which colonies have received the uncut plasmid and which have the plasmid with our enzymes, under the induction of IPTG, since the colonies with the uncut plasmids will be expressing RFP. A Golden Gate with our DeHa5 (BBa_K4868999) transformation plate can be seen in figure 4. The image was taken under the exposure of blue light, which makes it easier to distinguish between fluorescent and non-fluorescent colonies, however, with RFP it is also possible to see the red color without light excitation.

For optimal Golden Gate efficiency, we set up a reaction containing the following components:

Destination vector	75 ng
Insert	1:2 molar ratio (vector:insert)
10x T4 Ligase buffer	2 µL
T4 Ligase	0.5 µL
PaqCI activator	0.25 µL
PaqCI	0.5 µL
H20	Up to 20 µL 2

The cloning then consisted of 25 cycles of restriction at 37°C for 3 minutes, followed by ligation at 16°C for 4 minutes. After the 25 cycles the enzymes were heat inactivates at 60°C for 15 minutes.

Figure 4: Golden Gate of DeHa5 into pET-51b+ BBa_K4868001 exposed to blue light showing the fluorescent colonies. The fluorescent colonies have not received DeHa5, so by selecting the non-fluorescent colonies the likelihood of selecting a colony with correct insertioni of DeHa5 increases significantly.

For evaluation of the success of the Golden Gate cloning, a colony PCR was performed. The result can be seen in figure 5. Samples 1-6 are colonies that didn’t fluoresce, which means they most likely have DeHa5 inserted. Samples 7&8 are colonies that fluoresced, which shows that there will be no amplified region if there still is RFP in the plasmid. Samples 9&10 are plasmids that through sequencing have been proven to have DeHa5 inserted correctly, so they show where we expect the amplified DNA band of 1139 bp. Samples 11&12 are WT so they show that no genomic DNA is amplified. Sample 13 is a H2O and primer contamination control, which shows that neither of the primers, or the water that was used was contaminated.

Assessment of the Riboswitch

The functionality of the riboswitch was tested.

Assessment of GFP Fluorescence by FACS

An experiment was made to assess the response of the riboswitch at a range of different fluoride concentrations. This experiment was done using Fluorescence-activated Cell Sorting (FACS). For this experiment, NEB’s T7 Express Competent E. coli (NEB #C2566I) containing the pET-51b plasmid with the BBa_K4868001 part were grown at 37 ℃ ON in a 96-well plate containing 100 μg/mL Ampicillin and 0.4 mM IPTG as well as 0, 47, 84, and 112 mM NaF at 37 ℃. T7 Express Competent E. coli (NEB #C2566I) wildtype was grown in a 96-well plate with the same compound concentrations and conditions. Triplicates were made of each NaF concentration. Samples were diluted until 15000-25000 events/second were detected and water was run between samples. Gates were made to detect single cells.

Figure 6: Violin plots made from FACS data showing the distribution of GFP fluorescence from NEB’s T7 Express Competent E. coli (NEB #C2566I) cells that have been exposed to different concentrations of NaF.

Figure 7: Violin plots made from FACS data showing the distribution of GFP fluorescence from NEB’s T7 Express Competent E. coli (NEB #C2566I) cells containing BBa_K4868001 that have been exposed to different concentrations of NaF.

Figure 6 shows the same levels of GFP fluorescence in the WT cells at all concentrations of NaF used in the experiment. Cells containing a plasmid with BBa_K4868001 tend to fluoresce more when they are exposed to higher concentrations of NaF (Figure 7). This shows that the riboswitch is sensitive to different concentrations of NaF and that it is possible to use the riboswitch to detect different levels of fluoride. Cells containing the pET-51b plasmid with BBa_K4868001 also fluoresce more when exposed to no NaF than the wild-type cells do. This suggests that the fluoride riboswitch (BBa_K4868000) is leaky. From the same data, another plot was made showing the fluorescence of the most fluorescent cells in the samples (Figure 8).

Figure 8: Assessment of the top 1 % fluorescent cells from a sample containing NEB’s T7 Express Competent E. coli (NEB #C2566I) with BBa_K4868001(green) and without BBa_K4868001(blue). Compared to the WT, cells with BBa_K4868001 fluoresce more at all tested concentrations of NaF.

From this plot (Figure 8) the same tendency can be seen: The cells containing BBa_K4868001 fluoresce at all tested concentrations of NaF. Compared to this, the most fluorescent cells in the wild-type samples did not fluoresce at all.

Assessment of GFP Fluorescence by Fluorescence Microscopy

Another experiment was made to assess the sensitivity of the fluoride riboswitch (BBa_K4868000) that controls the GFP (BBa_K2541400) expression in the Rosetta E. coli strain. This was done using fluorescence microscopy. This was also tested at different NaF concentrations. To prepare the samples for fluorescence microscopy a culture of the Rosetta E. coli strain was incubated for 16 hours in LB media containing 200 μg/mL Ampicillin for 37 ℃. This culture was then diluted and incubated in tubes with NaF concentrations of 0, 50, 100, and 150 mM NaF and expression of both RFP and GFP was induced by a concentration of 0.4 mM IPTG. These cultures were incubated in the same conditions for 4 hours. The cells were subsequently fixed for examination by microscope.

Figure 9: Microscopy images of E. coli Rosetta showing GFP fluorescence. 100 mM of NaF produces the most fluorescent cells. Fluorescence is still present at 0 mM NaF.

From the images produced by fluorescence microscopy of the cells (Figure 9), it was noticeable that the cells fluoresced the most at 100 mM NaF. It was expected that there would be no fluorescence at 0 mM NaF since NaF is necessary for the activation of the riboswitch. This is not the case, most likely because of background expression of GFP. These results confirm the results from the previously mentioned FACS experiment.

Assessment of GFP Fluorescence by Plate Reader

One last experiment was performed to determine how the riboswitch responds to different fluoride concentrations. For this, a plate reader was used to measure GFP fluorescence on a 96-well plate. A culture of NEB’s T7 Express Competent E. coli (NEB #C2566I) containing the pET-51b plasmid with BBa_K4868001 was incubated for 16 hours at 37 ℃ with an Ampicillin concentration of 100 μg/mL. bacteria from this culture were distributed on a 96-well plate with a range of different NaF concentrations from 0 mM to 200 mM. This experiment confirmed the results from the FACS and the fluorescence microscopy regarding the level relative level of relative fluorescence produced in the presence of different levels of fluoride. The highest level of fluorescence was detected at 100 mM NaF. There can also be observed a decrease in fluorescence at concentrations above 100 mM which most likely is because the fluoride levels become too toxic so the cells have less expression activity. The wildtype T7 Express Competent E. coli (NEB #C2566I) shows close to no fluorescence at all concentrations of NaF (Figure 10).

Expression of RFP

This was tested to evaluate the expression of genes located in the insertion site of BBa_K4868001.

It was tested if it was possible to induce the expression of RFP in NEB’s T7 Express Competent E. coli (NEB #C2566I). This was tested by exposing a culture to 0.4 mM IPTG for 4 hours followed by normalization of cell count and subsequent cell lysis and loading onto a Tris-Glycine gel (Thermo Fisher Cat. No. XP00165BOX).

This process included growing the bacteria in a shaking incubator for 15 hours overnight at 37 ℃ followed by the production of two culture tubes with a 50x dilution of the previous culture. These cultures were incubated for another 3 hours under the same conditions as before. One of the culture tubes was then induced by 0.4 mM IPTG and both cultures were incubated for another four hours. This experiment used a concentration of 100 μg/mL Ampicillin in all cultures. The IPTG-induction was followed by normalization of cell count by OD600 and cell lysis by boiling in 1x SDS loading dye. These samples were then loaded onto a Tris-Glycine gel and an SDS-page gel electrophoresis was conducted at 120 V for ⁓ 90 minutes. The gel was then washed in demineralized water followed by staining by a Coomassie Blue staining solution for 5 minutes. This was followed by further washing.

Figure 11: Picture of SDS-page gel, showing the PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (ThermoFisher Cat. No. 26619) in the first well, the cell lysate control in the second well, and the cell lysate from the induced culture in the third well. The successful induction of RFP can be seen by the protein band around 25 kDa produced from the IPTG-induced sample, which is not visible on the control.

Figure 11 shows that RFP was successfully induced in the culture that was exposed to IPTG at a concentration of 0.4 mM. This can be seen by the protein band that is present in the induced sample at around 25 kDa which is not visible on the control sample.

References

Lee, J., Sung, S. E., Lee, J., Kang, J. Y., Lee, J. H., & Choi, B. S. (2021). Base‐pair opening dynamics study of fluoride riboswitch in the bacillus cereus crcb gene. International Journal of Molecular Sciences, 22(6), 1–12. https://doi.org/10.3390/ijms22063234 Thavarajah, W., Silverman, A. D., Verosloff, M. S., Kelley-Loughnane, N., Jewett, M. C., & Lucks, J. B. (2020). Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor. ACS Synthetic Biology, 9(1), 10–18. https://doi.org/10.1021/acssynbio.9b00347