iGEM 2023 — Team sdu-denmark

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# Notebook

# Revised Notebook

#### This is the revised and summarized laboratory book - - for more a more detailed description of our daily work, please scroll down to the daily notebooks.

# Weekly Updates

## May

**Week 18: Safety training**

On the 1st of May we received a laboratory safety course for class I laboratories at Micro. Our safety training included, among other things, a fire evacuation plan, use of proper safety equipment such as a lab coat, knowing when and when not to use gloves, and which kind to use. At the same time, proper hygiene like cleaning of the workspace, used equipment or oneself. An introduction to the use of, for example, chemical databases, and chemical hazard pictograms was also included in the safety course. More qualitatively, we were also introduced to the social working environment and the organizational diagram for department heads.

**Week 19 & 20: “Baby steps”**

**Micro:** Our routine training in the laboratory continued. We kept making stocks of things we would need, such as LB-media, a 50% glycerol stock for freeze stocks, and a Transformation storage buffer (TSB) to make competent cells. This week, we also did our first successful transformation, and tried our hand at colony-PCR.

**Week 21: "The First Successes"**

**Micro:** Our routine training in the laboratory continued. We kept making stocks of things we would need, such as LB-media, a 50% glycerol stock for freeze stocks, and a Transformation storage buffer (TSB) to make competent cells. This week, we also did our first successful transformation, and tried our hand at colony-PCR.

**TEK:** As a side project, we started making a more sodium fluoride (NaF) tolerant mutant of our *E. coli* Rosetta-strain on 96-well plates (hereafter referred to as ALE). We made NaF-stocks with concentrations of 640mM and 800mM by mixing NaF with LB-media. As a note, we did not include any antibiotics in the 96-well plate experiments, although our Rosetta-strain carries a resistance gene for chloramphenicol on a plasmid. For this reason, we also later discovered that our bacteria had lost their chloramphenicol resistance.

## June

**Week 22: "Exam stress"**

**Micro:** For the month of June, our iGEM-work continued solely at **TEK**.

**TEK:** We kept experimenting with the best concentration to use for our NaF-tolerance experiments. This week, we used a stock concentration of 800mM NaF. We had to reheat our NaF-stock as it is sparingly soluble in this concentration. This week we also began to make freeze stocks of our ALE, as “checkpoints” in our experiments.

**Week 23: "The Daily Groove"**

**TEK:** The routine evolution protocol continued. We took turns going into the lab, allowing each other to also read up on our curriculum for exams.

**Week 24: "Critical Thinking"**

**TEK:** After some trial-and-error with our sodium fluoride-concentrations, we realized that 800mM was too concentrated, and also needed to be reheated each day to be properly dissolved. We adjusted our experiments to use a concentration of 320mM NaF. This week, we also compared the original Rosetta strain to our ALE strain. Please see the results on our [Results page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

**Week 25: "Re-evaluations"**

**TEK:** The 96-well plate experiments did not yield the results that we had wished to see. The growth pattern of the bacteria was too irregular. Therefore, we decided to start another NaF-tolerance experiment series and run it parallel to the 96-well plate experiment. This time, our bacteria would be grown on LB-agar plates. We cast LB-agar plates with chloramphenicol (CML). Unfortunately, the bacteria had not grown on the plates. We realized that the ALE must have lost its CML-R plasmid because we did not use CML on the 96-well plates. Thus, we decided to start this experiment anew with the original Rosetta-strain – this time on LB-agar plates with CML (30 µg/mL). The mutant evolved from these experiments will be referred to as ALE2. We made plates ranging in concentrations from 0mM to 500mM NaF.

The way we cast the plates at this point was sub-optimal. We created one bottle of LB-agar and added CML. This bottle was reheated for each day we cast new plates – followed by the addition of a required amount of NaF. We quickly realized that this procedure would decrease the quality of the antibiotic. So, from here on out, we opted to, instead, reheat only the agar, and mix it with CML and NaF in 50mL Falcon-tubes for each.

**Week 26: "The Grind Continues"**

**TEK:** We continued to run the 96-well plate experiments alongside the new agar-experiments. At this point, the growth of our ALE2 was very irregular on the plates. One day, the ALE2 bacteria grew on a plate with a concentration of 280mM NaF, the next, we had to decrease the concentration to >130mM NaF to observe growth.

Because we ran out of our 320mM NaF stock solution to the 96-well plate experiments, we instead decided to use our 500mM NaF.

## July

**Week 27: “Two Steps Forward, One Step Back”**

**Micro:** We started out the week by transforming the *E. coli* Top10-strain with ampicillin (Amp) resistance followed by miniprep of the plasmid. We also ran a high-fidelity PCR on each of our DNA-pieces which encoded our dehalogenases and ran part of the PCR-product on a gel, to verify our DNA-pieces. We then saved the rest for later use. This week, our first Gibson assembly of our backbone plasmid and insertion piece took place. We attempted to transform this into the original Rosetta-strain and examined our results using colony-PCR.

This first attempt did not yield any useful results, so we repeated this workflow a few times over the course of the week. Our 3rd Gibson assembly was successful, so we purified our plasmid containing our insertion piece, and we could now attempt to insert our dehalogenases into our backbone plasmid with Golden Gate (GG) Cloning.

**TEK:** NaF-tolerance-development of ALE2 on agar-plates continued.

**Week 28: “New protocols and Reevaluations”**

**Micro:** We attempted the first GGC this week. None were successful, despite us adjusting DNA-concentrations and tweaking the protocol. We also wanted to test if our fluoride-sensitive riboswitch worked, so we plated out ALE1 with no plasmid, ALE1 containing the pET51b plasmid with RFP and the variants with genes encoding the DeHa’s. The agar plates contained different concentrations of NaF and kanamycin. Some also contained IPTG and ampicillin as well. We did this to discern if the kanamycin-resistance of ALE1 corresponded with the amount of NaF that was in the plates. Unfortunately, cells were kanamycin-resistant regardless of whether the plate contained NaF or not. This pointed to a leaky riboswitch. This meant we had a lot of reevaluations to do. Therefore, we designed four new DNA-parts to test out and ordered them.

**TEK:** This week, we decided to see if our 96-well experiments had been effective, by testing whether our ALE-strain grew better than the original strain in NaF. We made overnight cultures of every other freeze stock we had made and measured the OD over the next few days. The resulting graphs did not look as we expected them to, partially because some of the wells had dried out, so we decided to try again later. We had also forgotten to add CML to the wells.

**Week 29: “Ups and downs”**

**Micro:** After all the failed GG-reactions, we thought it would be a good idea to test the working stocks of our primers for contamination. We found that one of them was, indeed, contaminated, so we threw it out. We also decided to modify our GG-protocol so that rather than a “single-tube-reaction”, we separated the reaction into cutting of the DNA-pieces and ligation of the cut pieces, respectively. This time, the reaction worked! Now, we had a sample with, presumably, our backbone plasmid with one of our dehalogenases, DeHa2, inserted!

**TEK:** With our second attempt at comparing the OD of our ALE1 and original Rosetta-strain, we added CML to the wells of the 96-well plate and stored it in a tightly sealed bag to prevent the wells from drying out. The OD was measured, but a lot of the bacteria had accumulated at the bottom of the wells. Therefore, the plate was stored in the refrigerator overnight, and the next day, the bacteria were resuspended, and the OD was measured again.

We also wanted to check the NaF-tolerance of the ALE2-strain. To do this, we made a dilution row of ALE2 and the original Rosetta-strain, respectively. We plated them out on agar-plates with varying NaF-concentrations. We found that ALE2 was more tolerant of NaF than the original strain!

**Week 30: “Back to the Drawing Board!”**

**Micro:** This week was spent waiting for sequencing results from various DNA-pieces. Among those were the plasmid backbone + insertion piece from our successful “Gibson assembly 3” and our newly ligated plasmid backbone + DeHa2, both with our forward and reverse primer.

We also decided to redo the test of our riboswitch, this time more thoroughly. In summary, these results revealed that our kanamycin resistance was constitutively active. This meant that we had to re-design our plasmid. For more details on these results, please click [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

**TEK:** With the newly acquired evidence that our ALE2-strain experiments were effective, we decided to keep evolving them to see how much we could push the NaF-tolerance of the strain.

## August

**Week 31: “Sequences and Successes”**

**Micro:** For the first time, we had DNA-samples sequenced! The lucky winner of this honor was the DNA-sequence for our DeHa2. Unfortunately, though, the sequencing results were rather disappointing, and seemed to show a frame-shift mutation at the end of the DNA-product. However, we received a new and different miniprep kit, which yielded much higher DNA-concentrations than our previous one.

Lastly, over the weekend, we decided to test how much PFOA our cells could tolerate, by plating them out on agar plates with increasing PFOA-concentrations.

**TEK:** At this point, the ALE2-strain seemed to have reached a plateau in their NaF-tolerance. Furthermore, we had to focus all efforts on our main project, thus concluding the evolution of NaF-tolerance in *E. coli* Rosetta. This also concluded our work at TEK.

**Week 32: “New... group members?”**

**Micro:** Our group decided that it was rather difficult to keep track of all our new DNA-parts. None of them really rolled off the tongue. Therefore, we decided to name them. Introducing the newest (and arguably most important) wetlab-personalities: Anders, Ken, Ginny, and George! All but George had now successfully been cloned into our plasmid.

The rest of the week was spent GGC dehalogenases into our plasmids, running colony-PCRs and checking that everything was assembled correctly.

**Week 33: “The battle of plasmids begins.”**

**Micro:** Now that most of our plasmids were loaded with a DeHa-gene, came the time for our selection process. We began testing Anders, Ken and Ginny for both RFP and GFP-expression. Poor George was lagging behind a little, and still needed to be transformed into Rosetta.

Fluorescence microscopy revealed that our plasmid, Anders, seemed to have the clearest response to increasing NaF-concentrations.

Our supervisor, Lin, proposed that we should test Anders in both our NEB-cells and ALE1-strain. She theorized that the NaF-tolerance might impact the fluoride-sensitivity of the cells, so, once again we prepared our cells for fluorescence microscopy. Unfortunately, it seemed that Lin was right. This led us to wonder whether the same might be the case if we were to put Anders in ALE2. For good measure, we also wanted to see the fluorescence of Anders in the original Rosetta-strain, too. Thus, we started the process of transforming Anders into these two strains.

**Week 34: “Choosing our plasmid”**

**Micro:** We decided to test the riboswitch in Anders and Ginny respectively, as they contained two different ones. After reviewing the results, we settled on Anders as our final plasmid, as it fluoresced more consistently under different circumstances. Later, we sent in Anders to sequencing, both without and with various DeHa’s.

Over the years, many iGEM-teams at SDU have battled hard and bravely in numerous attempts to make calcium chloride competent cell stocks. Despite the words of warning from our supervisors, we decided to challenge history – and with great success! We made a batch of approximately 100 1mL tubes of competent Rosetta cells! Subsequently, we transformed Anders with DeHa 1, 2, 4 and 5, respectively, into the newly competent cells, followed by a colony-PCR. To conclude the week, we transformed Anders with DeHa’s into ALE2. We finished with a colony-PCR of these cells and made freeze stocks for future use.

**Week 35: “Fresher’s Flu and Access Quarrels”**

**Micro:** This week was rather challenging, with many people falling ill with fresher’s flu and security systems resetting for the start of the new semester. This meant that we had to retake lab safety courses for TEK, and therefore had a bit of a setback, timewise. This experience was especially sour, as we discovered this on a weekend after spending a whole day preparing samples and solutions for an experiment regarding optimization of IPTG-induction.

We were also seriously starting to investigate buying a fluoride probe to measure enzyme kinetics.

## September

**Week 36: “FACS, fluoride probes, and plan B’s”**

**Micro:** At this point we got into contact with sales employees, but we feared that shipping times might put a stick in the wheel of our project. This was definitely the time to think about plan B’s.

Speaking of plan B’s… as we were preparing to transform our error-prone dehalogenases, we found that Rosetta had a difficult time stomaching our plasmid. This, combined with previous results that indicated that the riboswitch might work better in other cells, sparked the idea that maybe it was time to try a new bacterial strain. But would we even have time for that…?

On a lighter note, we got to try our hand at FACS this week at Odense University hospital, because it was difficult to determine the most fluorescent colonies at the UV-Vis-plate at TEK. FACS seemed to be a better alternative for sorting cells based on fluorescence.

**Week 37: “Big decisions and Stubborn Strains”**

**Micro:** We decided to order a new strain after all. We went with T7 Express Competent *E. coli* (High Efficiency), referred to as “NEB T7”, from New England Biolabs. While waiting, we utilized time in the lab as well as we could. We spent time optimizing our Golden Gate protocol on another strain, BL-21, while we waited. What a workload! This was near impossible because BL-21 DID. NOT. WANT. TO. GROW!

**Weeek 38: "A welcome arrival"**

**Micro:** Finally, our new cell strain, NEB T7, arrived! This meant go-time! Luckily, these cells were receptive to our plasmid, and willing to grow for us! This meant that we were finally able to run induction experiments – we wanted to verify that our Dehalogenases were expressed, before lysing the cells. The SDS gods were against us, however, so we could not conclude anything from the gels that we ran.

**Week: 39: “Feelin’ the pressure”**

**Micro:** This week, we transformed our error-prone dehalogenases into the NEB T7 strain and started running FACS on them!

Still feeling a bit down from last week’s SDS-page results, we decided that a more practical approach was necessary. We had to French press our cells and purify our protein to check whether they were induced. Despite leaky equipment and low protein yields, the SDS-pages we ran, confirmed that DeHa4 and DeHa5 were indeed induced.

As a side note, we suspect that DeHa1 and DeHa2 were also induced, but might be in the solid phase of the protein purification kit. Due to time pressure, we decided to only proceed with DeHa4 and DeHa5. Unfortunately, none of the departments at our university had a fluoride probe, which we needed to determine enzyme kinetics. So, we ordered one!

## October

**Week 40: “iGEM projects can only be 80% done”**

**Micro:** This was a big week, as we selected for the best error-prone dehalogenases! This involved a whole-day intensive laboratory experiment, but oh man, did we feel accomplished!

Our work was not yet done, though; we also had to send in the 52 samples that we collected, to sequencing, so we would not end up testing duplicates in the end. Having the DNA-sequence would also allow us to predict the tertiary protein structures. Furthermore, we received the results from our whole-genome sequencing of ALE1 and ALE2 strains. Now we could finally deduce where the increased fluoride tolerance came from!

# Daily Notebook from Micro

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# Daily Notebook from TEK

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# Engineering

* [Overview](#overview)
* [Insertion Piece Iteration 1](#insertion-piece)
* [Insertion Piece Iteration 2](#iteration2ip)
* [Fluoride Tolerance Iteration 1](#ALE)
* [Fluoride Tolerance Iteration 2](#iteration2ale)

# Overview

Every project starts with an idea. To bring the synthetic biology idea to life, a systematic approach is required. The process an engineer goes through in the development of a product can be divided into iterative stages. Each iteration will improve the system, based on knowledge gained from previous iterations. The four stages are **Design, Build, Test, and Learn.**

**The Design stage** is the first step in the engineering cycle. Firstly, the desired function should be defined. Secondly, a conceptual draft should be sketched, so that it can be questioned, tested, and simulated using various computational tools. After finding a successful design that, on paper, achieves the desired function, it is time to move on to the build stage.

**The Build stage** is where the designed construct is transformed into an organism of choice. The methods of assembly used in the build stage should consider the design specifics. This stage also encompasses the experimental methods in the first set of cycles discussed.

In **the Test stage**, the function of the modified organism, or the assembled construct inside the chassis, is evaluated.

Lastly in **the Learn stage**, the results from the test stage are used to improve the design. The initial design is rarely sufficient, so new ideas for the design are generated - hence the cyclic nature of the process. After adequate iterations, a useful design should succeed.

![engineering-cycle-img](https://static.igem.wiki/teams/4868/wiki/engineering-page/engineering-cycle.svg)

**Fig. 1:** The stages of the engineering cycle.

**Our aim in the laboratory is to successfully degrade PFAS** in water samples, and then later be able to utilize our solution to clean drinking and wastewater at large scale. During our project, we went through the engineering cycle multiple times. Below you can see our work optimizing our dehalogenases (DeHa) to better degrade PFAS, using PFOA as a model molecule. You can also read about our work making *E. coli* Rosetta more fluoride tolerant.

# Engineering success on the DNA construct insertion piece

The specifics of how our final designed construct works and how it is assembled are described in greater detail on the [Parts Registry](http://parts.igem.org/Part%3ABBa_K4868001) page.

## Iteration 1 – *Initial experimental design*

### Design

We designed a sequence where the dehalogenases (DeHa) could be expressed in a system, where **higher protein efficiency results in higher antibiotic resistance.** Kanamycin was the antibiotic we chose as a selection marker. See figure 2 for the sequence.

The initial construct was designed with a site occupied by an RFP (red fluorescent protein) gene. This gene would allow for differentiation between colonies that have been transformed and those that have not. This RFP site was a placeholder for the DeHa gene. **The DeHa gene would be assembled by Golden Gate Cloning using PaqCI restriction enzyme**, removing the RFP gene.

![first-iteration-with-color-crop-01](https://static.igem.wiki/teams/4868/wiki/engineering-page/first-iteration-with-color-crop-01.svg)

**Fig. 2:** The first iteration of the insertion piece. It consists of two major parts: an insertion site and a fluoride sensitive riboswitch. The insertion site is flanked by PaqCI recognition sites that can be substituted by the DeHa genes. The fluoride riboswitch is associated with a kanamycin resistance gene. Increasing concentrations of fluoride from DeHa enzyme activity results in higher resistance. The insertion piece has a size of ~2000bp.

The construct contains a riboswitch associated with inducible kanamycin resistance. **The riboswitch becomes active when bound by fluoride**.

The assembly was simulated in Benchling and found to be successful.

We tried ordering our insertion piece in one part, but it was not able to be synthesized. Therefore, from now on, we ordered the insertion piece in two parts: the insertion sequence and the riboswitch part. This improved the quality and success of synthesis. The insertion piece is then made up of the two pieces, and then combined with Gibson assembly when inserted into the plasmid.

### Build

Using Gibson assembly, the insertion sequence was cloned into the pET-51b(+) expression vector and transformed into NEB competent *E. coli*. **The DeHa gene was inserted using Golden Gate Cloning.** Later, **the plasmid was purified and transformed into our ALE1 Rosetta strain** from our fluoride tolerance experiments.

### Test

To confirm the successful integration of our DeHa genes into [BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001), our insertion piece, we ran gel electrophoresis.

To test whether the riboswitch worked, we investigated the ability of the ALE1 strain to grow on plates with kanamycin, sodium fluoride (NaF), and Isopropyl β- d-1-thiogalactopyranoside (IPTG).

**The expected results would be growth only when NaF was present, as this would result in expression of kanamycin resistance.** It was found that there indeed was growth, but we also found growth with no NaF present, which was not expected.

### Learn

We verified the correct assembly of [BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001) and the DeHas since the size of the bands matched the size of [BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001), as seen on figure 3a, and the DeHa genes, as seen on figure 3b.

![3a3b-gel](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/3a3b-gel.svg)

**Fig. 3:** 3A: Electrophoresis gel results from a colony-PCR after Gibson assembly of pET-51b and the designed insertion piece. As expected, the resulting DNA segments have a length of ~2000bp. 3B: Electrophoresis gel results from the insertion of Deha 1 (720bp), Deha 2 (675bp), Deha 4 (900 bp) and Deha 5 (672 bp).

We learned that it was **difficult to differentiate some of the dehalogenases from RFP** on the gel. This is because the RFP genes had a length of 675 bp. The genes encoding the DeHa enzymes range from 672 bp to 900 bp and therefore the bands on the gel from the DeHas and the RFP would be too close together to distinguish from each other.

We found that the bacteria were resistant to kanamycin when exposed to NaF, even though there was no riboswitch activation. This indicated **a leaky riboswitch.**

We also learned that using **kanamycin resistance as a means of selection was suboptimal.** It would be hard to determine whenever the bacteria were resistant against Kanamycin because of the riboswitch activation, or because the riboswitch was leaky. This would make it difficult to select the most effective enzyme since it was not possible to determine what caused the resistance.

## Iteration 2 – *Introduction of GFP*

### Design

After the previous iteration, multiple changes had to occur. Firstly, the selection marker associated with the riboswitch had to be **changed from kanamycin resistance.** Secondly, the riboswitch had to be reinvestigated to ensure proper activation.

For the selection marker, we decided to opt for **super-folder green fluorescent protein (sfGFP)**, as this would allow for quantitative selection of the colonies.

We reevaluated the genetic elements surrounding the riboswitch, as they may be essential to the function. We found that in the paper *“Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor”* by Thavarajah et al., they described the riboswitch1. Here they used a specific constitutive promoter, the Anderson Promoter ([BBa\_J23119\_Spe1](http://parts.igem.org/Part%3ABBa_J23119)). **We replaced the T7 promoter with an Anderson promoter to ensure the riboswitch would only be active when induced by fluoride.**

Our insertion piece was further modified. From the Thavarajah et al. paper we also inserted the T1 terminator. We called this insertion piece **[BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001).**  See the modified insertion piece in figure 4.

#### Iteration 1:

![first-iteration-with-color-crop-01](https://static.igem.wiki/teams/4868/wiki/engineering-page/first-iteration-with-color-crop-01.svg)

#### Iteration 2:

![second-iteration-with-color-crop-01](https://static.igem.wiki/teams/4868/wiki/engineering-page/second-iteration-with-color-crop-01.svg)

**Fig. 4:** A comparison of the first and second iteration of the insertion piece. The major differences lie in the marker gene being changed from kanamycin resistance to sfGFP, and the promoter for the marker gene being substituted by the Anderson promoter.

### Build

We cloned the new solution **[BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001)**, into the pET-51b(+) expression vector using Gibson assembly. **The assembled plasmids were then transformed into NEB competent cells.** Following this procedure, the plasmids were purified and introduced into the ALE1 Rosetta strain from our fluoride tolerant experiments and the original *E. coli* Rosetta strain.

### Test

For the first test of the riboswitch, fluorescence microscopy was performed. We did this to test the fluorescence response of **[BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001)** to different concentrations of NaF.

The test was performed where the original Rosetta, ALE1 strains, and NEB competent stable cells were incubated overnight. The following day the bacterial cultures were grown in tubes with Ampicillin (100 mM), IPTG (0.4 mM), and varying amounts of NaF. The cultures were then fixated and examined under a fluorescence microscope to investigate which concentrations of NaF resulted in the highest sfGFP detection. The results can be seen in figure 5.

![](https://static.igem.wiki/teams/4868/wiki/engineering-page/iteration2-insertion-piece.png)

**Fig. 5:** Fluorescence microscopy experiment to examine which concentration of NaF (0 mM, 50 mM, 100 mM or 150 mM) would result in the highest sfGFP detection in the three strains ALE1, NEB competent cells, and original E. coli Rosetta.

In addition to fluorescence microscopy, the samples were added to two 96-well plates with Ampicillin and varying amounts of NaF: one plate for OD600 measurements and one for fluorescence measurement. The samples in the 96-well plates were measured in a **plate reader**, as seen on figure 6, and **Fluorescence-activated Cell Sorting (FACS) machine** seen on figure 7. The first results obtained showed a fluorescence optimum at 100 mM NaF.

Further experiments were then conducted to narrow down the exact amount of NaF needed to be added for optimal effect. The added NaF to cultures were of the concentrations: 75 mM, 85 mM, 95 mM, 105 mM, 115 mM, and 125 mM.

The riboswitch was also tested using FACS and a microscope. At the FACS there was sometimes detected 2 population, 1 population showing fluorescence and 1 population without fluorescence. This indicates that the **plasmid might be lost in some of the cells**, which might be due to unstable ampicillin that loses efficiency after too many hours in liquid at 27-37°C. Upon investigating the samples under the fluorescence microscope, it was confirmed that some of the cells had lost their pladsmid, since they showed no fluorescence.

![](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/plate-reader-test.png)
![](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/facs-test.png)

**Fig. 6:** The plate reader experiment testing the level of fluorescence produced with various genes inserted into the insertion site of RGFr. The graph shows sfGFP normalized fluorescence against fluoride ion concentrations. It can be seen for RGFr that the riboswitch is more sensitive when RFP is inserted at the insertion site. The NEB wild type shows the least amount of fluorescence.
**Fig. 7:** Assessment of the sfGFP fluorescence level produced by the 1 % most fluorescent cells using FACS. Each sample has a different DeHa gene inserted into the insertion site of RGFr except the NEB wildtype. The highest amount of sfGFP fluorescence can be seen from the cells containing DeHa 2 gene at the insertion site and the least amount of fluorescence can be seen from the NEB wildtype.

### Learn

As seen in figure 5 the ALE1 strain did not fluoresce noticeably, **no matter how high a concentration of fluoride** it was exposed to. This was unfortunate since this indicated that **the riboswitch wasn’t working**. One explanation for the fluoride tolerance could be that the bacteria had **improved their fluoride ion excretion**. The fluoride ions could thereby not activate the riboswitch, which would result in the cells **not fluorescing**. We were excited to see that this was **not the case for the original Rosetta strain and the NEB competent cells**. This verified that the fluoride ions usually enter the cell, thereby activating the riboswitch.

FACS and plate reader results from figure 6 and 7 show that some of the DeHas first release fluorescence detectable at a NaF concentration higher than 50 mM. This indicates that the riboswitch has a low sensitivity, which is a problem since we in the lab can only dissolve PFOA at a 1.2 mM.

# Fluoride Tolerance

## Iteration 1 – *Initial experimental design*

### Design

To accommodate the **fluoride-toxicity challenge** from the beginning of our project, we designed an adaptive laboratory evolution (ALE) experiment to confer a higher fluoride tolerance to our original *E. coli* Rosetta strain. We planned on inserting our pET-51b(+) expression vector with one of the DeHa genes, into this ALE strain.

For this purpose, we decided to use **increasing concentrations of fluoride (using dissolved sodium fluoride (NaF)) and UV light to increase the fluoride tolerance of the bacteria.** We decided that a 96-well plate would be suitable for this experiment because it allowed us to easily make replicates and dilution series of different NaF concentrations and the different lengths of UV exposure. We used a plate reader to measure the OD600 of the 96-well plates to provide insights into the bacterial growth efficacy of the different samples.

The original *E. coil* strain contained a plasmid with chloramphenicol resistance. We did not include chloramphenicol in the media to reduce growth inhibiting factors in the ALE experiment.

### Build

Because there is no DNA construct designed to transform into the chassis, this experiment did not have a build phase.

### Test

Each day, a new 96-well plate with varying NaF concentrations and bacteria selected from the most promising well of the 96-well plate experiment conducted the previous day. was incubated and OD600 was measured. The bacteria from the most promising well were then selected and used for further evolution.

The testing stage was repeated every day for a month.

To conclude the series of experiments, **we tested whether there was a difference in fluoride tolerance between the original *E. coli* Rosetta strain and the Rosetta strain** developed using ALE (from now on referred to as ALE1) in liquid media. In figures 8 and 9 the results from the comparison of original Rosetta and ALE1 can be seen.

![](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/ale1-compared-to-original.png)

**Fig. 8:** Comparison of the E. Coli Rosetta ALE1 strain that was developed in 96-well plates with the original E. Coli Rosetta strain. The graph depicts the mean OD600 of quadruplicates at each NaF concentration of each strain. There is no significant difference that can be seen, since the ALE 1 strain since the growth still is significantly inhibited. The lethal NaF concentration, however, seems to be higher for the ALE1 strain, so it has developed an increased tolerance.

![](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/original-rosetta-and-ale-1-petri.svg)

**Fig. 9:** Comparison experiment of the original Rosetta strain and ALE1. Both strains grew the same at the different concentrations of NaF. For the rest of the results click [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

### Learn

The Rosetta strain had chloramphenicol resistance encoded on a plasmid at the beginning of our experiments. We learned that after a month of not having been exposed to chloramphenicol, **the bacteria had lost the plasmid.** Therefore, they were not resistant to chloramphenicol anymore. This was a problem because we wanted to select our bacteria, making sure they were not contaminated. This problem was fixed by purifying the plasmid from the original Rosetta strain and then reintroducing it into the ALE1 strain.

From the experiments performed in liquid media, a difference between the two strains is observed. The ALE1 strain survives in concentrations >225 mM NaF while the original Rosetta does not. In contrast to the liquid media, no difference was seen between the two strains when grown on agar plates containing NaF.

## Iteration 2 – *Assisted evolution on agar plates*

### Design

We decided to reevaluate our experimental design due to the complications from the previous iteration.

For this version of the ALE experiment, we exchanged the 96-well plate for LB agar plates. This would, however, limit the number of different concentrations we could test every day. Furthermore, we were limited by the level of precision in our laboratory scale since we had to measure NaF on a daily basis. The NaF concentration needed the next day was based on the results of the previous day. We hoped that this approach would yield more consistent results.

We started this experiment while still trying to make ALE 1 work. Therefore, we decided it would be best to start over with the original *E. coli* Rosetta strain, rather than trying to evolve ALE1 further due to the loss of chloramphenicol resistance.

### Build

Because there is no DNA construct designed to transform into the chassis, this experiment did not have a build phase.

### Test

The ability of the bacteria to grow on different concentrations of NaF was assessed by looking at the plates after incubating. **The plate with the highest concentration of NaF that contained bacterial growth would be chosen and the bacteria would be radiated with UV light, resuspended, and plated on a new NaF containing agar plate.**

Agar plates were made with chloramphenicol and different concentrations of NaF. The first time this was done, bacteria from an *E. coli* Rosetta overnight culture were used, but as the experiment went on, **bacteria from the plate with the highest concentration of NaF that contained bacterial growth were used.** When the plates were dry, they were exposed to UV light, sealed with parafilm, and left to incubate overnight.

After incubation, the plates were assessed and the plate containing the most colonies would be used for the next plates.

When the development reached a plateau, it was determined in an experiment whether the resulting strain from this experiment (which will be referred to as ALE2) was more fluoride tolerant than the original Rosetta strain and the ALE1 strain.

![](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/original-rosetta-and-ale-2-petri.svg)

![](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/ale1vsale2.png)

**Fig. 10:** comparison experiment of ALE2 and original Rosetta strain, and ALE1 and ALE2. A comparison of ALE2 and the original Rosetta strain showed that ALE2 grew better than the original Rosetta strain at 200 mM NaF. A comparison experiment for ALE2 and ALE1 showed that ALE2 grew better than ALE1 at 200 mM NaF. For the rest of the results click [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

### Learn

**When examining the plates, it appeared as if the evolved ALE2 strain had improved fluoride tolerance.** Comparison between the ALE2 strain to both the ALE1 strain and the Rosetta strain showed, that the ALE2 strain was more tolerant to fluoride than both the ALE1 strain and the original Rosetta strain which can be seen in figure 10. Here **ALE2 grew on higher concentrations of NaF than ALE1 and the original Rosetta strain.**

We used ALE1 and ALE2 for multiple of our other experiments but found that the fluoride tolerance for ALE1 and ALE2 was probably due to the bacteria **pumping the fluoride out** and therefore making our riboswitch inactive.

*If you are interested in the Whole Genome Sequencing results for the ALE1 and ALE2 strains, please contact: sofielmoebes@gmail.com*

## Human Practices

[![next-page-button](https://static.igem.wiki/teams/4868/wiki/wiki-images/next-page.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)

## References

1. 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

{% endblock %}

{% extends "layout-new.html" %}
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# Experimental Design

* [Overview](#overviewdesign)
* [Methods](#methods)
* [Detailed Methods](#methods-detailed)

# The Design of our DNA Construct

Given the problem with PFAS contaminated water, we strived to eliminate PFOA (one of the most widespread PFAS) from water supplies.

Since we are working on optimizing a set of dehalogenases, we need a system that can **allow us to select the most efficient bacteria**. Our part [BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001) is a composite part consisting of two parts:

1. **A fluoride-sensitive riboswitch**, associated with a green fluorescent protein.
2. **An insertion site**, where the dehalogenase genes can be inserted in a modular way.

When the **dehalogenases degrade PFOA** and cleave the C-F bonds, **fluoride is released**. This will **activate the riboswitch**, resulting in the expression of the **green fluorescent protein**. The more effective the dehalogenases are at degrading PFOA, the more fluoride will be released, and the more the cells will fluoresce. The principle of the selection system can be seen on figure 1, and the basic mechanism of a riboswitch can be seen on figure 2.

To insert the dehalogenases after the DNA construct has been assembled in a backbone, we use the highly efficient Golden Gate Cloning method. The site where the dehalogenase will be inserted is **occupied by a red fluorescent protein gene**, which is replaced during the Golden Gate Cloning. Colonies that do not **have the dehalogenase gene inserted** will fluoresce red and can therefore be **easily detected**.

![](https://static.igem.wiki/teams/4868/wiki/design-page/iptg-induction-system-2.svg)

**Fig. 1:** Schematic of the mechanism of our system. IPTG induces expression of DeHa. DeHa will cleave C-F bond in PFOA, which releases free fluoride. The fluoride will active the riboswitch which induces expression of the marker gene, GFP. GFP is then measured to quantify the effectiveness of the given DeHa.

![rib-wo-f](https://static.igem.wiki/teams/4868/wiki/design-page/riboswitch-wo-fluoride-2.svg)
![rib-w-f](https://static.igem.wiki/teams/4868/wiki/design-page/riboswitch-w-fluoride-2.svg)

**Fig. 2:** Schematic of the basic principle of a riboswitch. Notice how mRNA is transcribed without the presence of fluoride. It forms a hairpin structure, blocking translation. Upon addition of fluoride, a conformational change occurs, which opens the hairpin to a looser structure, allowing translation.

# Methods Used in the Laboratory

The design we chose shaped the methods we used in the project. Here are short descriptions of what methods we used and why. Detailed descriptions of these methods can be found in the dropdown “Method Details” at the bottom of the page.

#### Construction of plasmid

**Plasmid assembly:** To construct a plasmid for transformation, we used **Gibson Assembly**. Due to problems with synthesizing our insertion piece, we had to have it **synthesized in two parts**. Gibson Assembly is useful for **inserting multiple fragments at once** into a backbone.

**Insertion of dehalogenases:** The dehalogenase genes needed to be inserted into the plasmid after assembly, in an efficient manner. For this, we chose the **Golden Gate Cloning** technique, where we utilized the PaqCI type IIS restriction enzyme.

#### Creation of mutants

**Mutagenesis:** To create a huge library of mutants, as a means of creating better dehalogenases, we opted for untargeted mutagenesis with **Error-prone PCR.**

**Selection of optimized enzymes:** After creating an array of mutants, we needed a way to select the most efficient bacteria. Given the correlation between fluorescence and degradation efficiency, we decided to **use FACS to sort the most fluorescent cells**.

#### Extraction and evaluation of the dehalogenases

**Protein purification:** Since we wanted our final product to be purified enzymes, we used the **HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit**. The purification was verified with **SDS-Page**.

**Measuring efficiency:** It was essential to evaluate the efficiency and kinetics of our purified enzymes, and for this we used **F-NMR and a fluoride probe**, to measure the **release of fluoride** during the degradation.

#### Creating fluoride tolerant *E. coli*

Initially in our project, we wanted to create a strain of *E. coli* with **an increased tolerance to fluoride**. This would be beneficial in the experimental procedures generating more efficient dehalogenases, where **the bacteria could be exposed to high concentrations of fluoride** from the degradation process. Though we did succeed, we later learned that the fluoride tolerance would not be beneficial in our project, as described on the [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) page.

To create this tolerant strain, we used **Assisted Laboratory Evolution (ALE)**, where a **selection pressure of increasing fluoride concentrations**, and a **mutagen**, are applied **to accelerate adaptation** towards higher tolerance.

# Detailed Methods

#### Plasmid construction using Gibson assembly

We used Gibson assembly to insert the two parts of our insertion piece into our plasmid backbone, which, before the assembly, was cut and linearized with the restriction enzymes SgrAI and HindIII.

For the Gibson assembly to be successful, we added 30 base pair overhangs to both sides of both parts of the insertion piece. The added overhangs are identical to the DNA parts where the assembly will be taking place. This way the 5' exonuclease will create single-stranded overhangs that will be complementary and seamlessly assemble our insertion piece into our plasmid backbone.

![gib-assembly](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/long-gibson-assembly-1.svg)

**Fig. 3:** Overview of Gibson assembly used for plasmid construction. The single-strand DNA overhangs created by SgrAI and HindIII are not complementary, thus preventing the plasmid backbone from reassembling. The overhangs in the backbone do, however, match those in the insertion piece, allowing the integration of these DNA-sequences into the backbone.

#### Insertion of the dehalogenases with Golden Gate Cloning

![golden](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/golden-gate-cloning.svg)

**Fig. 4:** Principle of Golden Gate cloning. Golden Gate cloning includes, firstly, the cutting and removal of RFP out of the insertion piece, residing in the backbone. Like in Gibson assembly, the cut plasmid is unable to re-assemble. Next, the ligation step occurs, which integrates the DNA-sequence of the dehalogenase of interest into the insertion piece.

Golden Gate Cloning is a method for assembling DNA fragments. The method utilizes the IIS-restriction enzymes, where we specifically used PaqCI. Two or more DNA fragments flanked with compatible recognition sites can then be digested and seamlessly ligated. Because the product will no longer contain the original recognition site, there will be no re-digestion, driving the process towards 100% completion.

Golden gate cloning was chosen since the design would allow the red fluorescent protein to be cut out, and the DeHa enzymes to be inserted in its place. Our Golden Gate insertion site is controlled by the T7 promoter, which means that under the presence of IPTG, the colonies will either turn red or white depending on whether the cloning was successful. In this way, we also ensured that the colonies we selected for further analysis had received the DeHa, since it is not possible for the plasmid to re-ligate itself without DeHa, because of the overhangs we designed1. Figure 4 gives an overview of the RFP-dehalogenase exchange that happens in Golden Gate cloning.

#### Mutagenesis with Error-prone PCR

Since there were several unknown factors about the enzymes, it would be difficult to make targeted modifications. This is why we are using random mutagenesis instead. Error-prone PCR was chosen to create DeHa mutant libraries.

This approach was chosen because, unlike using in vivo mutation of the DeHa gene to generate diverse isotypes, this approach prevents mutations in the host metabolism and the riboswitch-selection system. This means we can be sure that improved PFOA degradation will be caused by a more efficient dehalogenase and not be a mutation in a different gene. This is important because the enzymes were purified following the selection process and any mutations outside the DeHa sequence would not improve the PFOA degradation in vitro.

The error-prone PCR kit from Jena Bioscience was used, which included PCR components that increase the mutation frequency of the DNA polymerase by 0.6-2.0%2. These PCR components included enhanced concentrations of magnesium ions and unbalanced levels of dNTPs. Using a Taq-polymerase, which does not proofread, increased the error rate.

Performing Error-prone PCR on the DeHa sequences isolated will ensure that there are several different sequences produced which all can be transcribed and translated to different enzymes. A schematic of an error-prone PCR reaction can be seen on figure 5.

![error-prone](https://static.igem.wiki/teams/4868/wiki/illustrations-images/design/error-prone-pcr-dna.svg)

**Fig. 5:** Simplified schematic of an error-prone PCR reaction. As indicated in the figure, an error-prone PCR reaction works the same way as a normal PCR reaction, amplifying the desired DNA-product. Using the specified kit mentioned in the text above, the mutation rate is 1/10,000.

#### Selection of optimized enzymes with FACS

Fluorescence-activated Cell Sorting (FACS) is a type of flow cytometry, which uses the scattering of light or fluorescence activity to sort cells. An illustration of this principle can be seen in figure 6.

![facs](https://static.igem.wiki/teams/4868/wiki/illustrations-images/design/facs.svg)

**Fig. 6:** Schematic overview of fluorescence-activated cell sorting (FACS). Bacteria are sorted by measuring the fluorescence of one single cell at a time. The machine can then sort the cells into different populations, e.g., low-fluorescence cells (orange) and high-fluorescence cells (green).

FACS was chosen as it is a high-throughput way to screen the DeHa libraries, which can easily contain several thousands of different enzyme variants.

After sorting the most fluorescent cells into individual wells on 96-well plates, the cells were incubated to initiate cell division. After 24 hours of incubation, the OD600 and fluorescence were measured which made it possible to select the most fluorescent bacterial lines among the variants. From the selected bacterial lines, the plasmid containing the improved dehalogenase enzyme was purified and submitted for sequencing.

After sequencing results had been assessed to make sure that the sequences differed from the original sequence and each other, the bacterial strains containing the plasmids were cultivated at a larger scale and the proteins were purified from them and tested.

#### Protein purification with the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit and SDS-Page

The resin used in this kit consisted of cross-linked agarose beads and had a protein binding capacity of up to 100 mg/mL3. The used kit has the article no. 80101.

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-page) is a method used to separate complex mixtures of protein. The first step is the denaturation of the proteins with an anionic detergent, which also binds to the protein, giving them a negative charge proportional to the protein's molecular mass. This is followed by electrophoresis, where the porous acryl gel matrix separates the proteins according to their size. The predicted result of an SDS-page resulting from the purified dehalogenases DeHa 1, 2 4, and 5 can be seen on figure 7.

![SDS](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/sds-page-of-deha.svg)

**Fig. 7:** Schematic representation of an SDS-page. The illustration represents the expected SDS-page resulting from purified protein samples of DeHa 1, 2, 4, and 5, respectively. The DeHa 1, 2 and 5 are expected to show bands at 25 kDa, whereas DeHa 4 has a size of approximately 35 kDa. The protein ladder used for size reference, also used in the laboratory experiments, is the PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Cat. No. 26619).

#### Measuring efficiency with F-NMR and a fluoride probe

We chose to use F-NMR since fluoride has a spin. It is possible to measure how the PFOA molecules are cut by the enzyme without the enzyme interfering with the measurements (because the enzymes do not contain Fluorine). F-NMR was used to determine if and how fast the enzymes work.

![F-NMR](https://static.igem.wiki/teams/4868/wiki/illustrations-images/design/f-nmr-of-pfoa.svg)

**Fig. 8:** 19F nuclear magnetic resonance spectroscopy image of PFOA.

Firstly, F-NMR is used to determine whether the enzyme cleaved the F-C and C-C bonds. This can be seen if the peaks in the spectra have shifted or disappeared. Afterward, F-NMR was used to study the kinetics of the enzymes, by taking an F-NMR spectra at regular intervals and comparing the spectra and see how the peaks “move around” in the spectra.

Fluorine-19 nuclear magnetic resonance spectroscopy (F-NMR) is a method typically used for structural analysis of molecules. This method takes advantage of the fact that certain nuclei have a spin. A Spin depends on the isotope of the element. When applying an external magnetic field, the nuclei will align with or against the field. Then a radio frequency is applied that makes the nuclei temporarily shift alignment. When the radio frequency is turned off, the nuclei emit energy which is detected, and results in an interpretable spectrum4. An example of such a spectrum can be seen in figure 8.

One of the final planned experiments of this project was to measure the enzymatic activity of the variant dehalogenases and compare it to the original dehalogenases, using an ion-sensitive electrode. Unfortunately, due to delivery issues, it was not possible to obtain the electrode before The Grand Jamboree. It would have been a valuable tool for determining the enzymatic activity of both the original and variant dehalogenases. An ion-sensitive electrode measures the electric potential of a solution, which results from the presence of ions. By measuring the electric potential of stock solutions with known fluoride concentrations, a reference curve could be created. With this reference curve, the electric potential of the solution can be converted to the concentration of fluoride.

![probe](https://static.igem.wiki/teams/4868/wiki/illustrations-images/design/probe.svg)

**Fig. 9:** Illustration of an ion-sensitive electrode.

In essence, the planned experiment involved creating several PFOA solutions corresponding to the number of dehalogenases to be tested. All would have the same PFOA concentration. Next, the desired enzyme would be added to its respective beaker. PFOA degradation using the dehalogenases of interest yields free fluoride ions, which would affect the electric potential of the solution. The electrode would then be used to measure the electric potential of the solutions over time, at set intervals. Using the reference curve, the data points could be converted to the fluoride concentration of the respective solutions at a given time. Thereby, the change in fluoride concentration over time could be calculated, and from this, enzymatic activity would be derived.

In conclusion, **an ion-sensitive electrode would allow for the measurement of the enzymatic activity of both the original and variant dehalogenases**. This would reveal whether optimization of dehalogenases using error-prone PCR is feasible.

For further information on how this experiment would have been conducted, please refer to SOP 30.

# Experiments For Our Plasmid

* Designed DNA & Plasmid
  + Gibson Assembly
    - Transformation into competent cells
      * Colony PCR
        + Plasmid Purification
          - Error-prone PCR
            * Golden Gate with mutated DeHa
              + Transformation into competent expression strain
                - FACS selection
                  * Plate reader selection
                    + Plasmid Purification & Sequencing
                    + Protein Purification
                      - SDS-page
                        * F-NMR
                          + Fluoride probe
          - Golden gate with original DeHa
            * Transformation into competent cells
              + Colony PCR
                - Purified Plasmid
                  * Transformation into competent expression strain
                    + Protein Purification
                      - SDS-page
                        * F-NMR
                          + Fluoride probe
                    + Riboswitch test
                      - Agar plates
                      - Microscope
                      - FACS
                      - Plate reader

# Fluoride Tolerance

* *E. Coli* Rosetta
  + ALE1
    - Transformation of plasmid with insertion piece
      * Riboswitch test
        + Agar plates
        + Microscope
        + Plate reader
  + ALE2
    - Transformation of plasmid with insertion piece
      * Riboswitch
        + Microscope
        + Plate reader

# Designed DNA & Plasmid

The DNA parts include the pET-51b plasmid and an insertion piece with a fluoride-activated riboswitch which is coupled to GFP. GFP will thereby be the selection tool for the most effective PFOA-degrading enzymes. The insertion piece also includes an insertion site for the dehalogenases. The insertion site has RFP as a placeholder and selection marker. It also contains PaqCI sites that match the PaqCI sites designed on the genes encoding the dehalogenases. This way, each respective dehalogenase can replace RFP, using Golden Gate cloning. This means that RFP can function as a second selection marker for successful Golden Gate cloning. Because the insertion piece is too long to synthesize, it had to be ordered in two separate parts. This made Gibson assembly an obvious choice for plasmid assembly. The final plasmid design has the part number [BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001). For a visual representation of our insertion piece, please visit our [project description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D).

# Gibson Assembly

Gibson assembly is a method that allows two separate pieces of DNA to be ligated and inserted into a plasmid. Therefore, it was used to couple the two segments of the insertion piece to the pET-51b backbone. The backbone had previously been cut with the restriction enzymes HindIII and SgrAI, giving it overhangs, which matched those on the insertion piece.

For more details on Gibson assembly, please see SOP 9.

# Transformation into competent cells from Gibson assembly kit

In this step, the product from the Gibson assembly is transformed into NEB(®) 5-alpha Competent *E. Coli* cells (Cat. No. NEB #C2987), referred to as NEB C2987. This was done with a heat-shock transformation, before being plated onto selection plates with 100 μg/mL ampicillin. The NEB competent cells do not encode ampicillin resistance on their genome, but pET-51b does. Therefore, ampicillin was used as a negative selection marker.

For more details on the used heat-shock protocol, please see SOP 13.

# Colony PCR

Colony-PCR is a method used to verify that the plasmid is assembled correctly by using primers that bind to short sequences in the backbone which flank the insertion piece. Therefore, a colony PCR from a successful Gibson assembly will reveal a band with the same length as the insertion piece, when run on a gel, which is roughly 2000 bp. Colony PCR was usually done on 8 colonies per transformation, followed by a gel electrophoresis. Two of the colonies that contained the insertion piece were selected, cultured, and saved in a freeze stock. *An overview of a colony-PCR workflow can be seen on figure 9.*

For a more detailed description of how colony-PCR was run, please see SOP 15.

![colony-pcr](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/colony-pcr-1.svg)

**Fig. 9:** Overview of colony-PCR workflow. From a plate of successful mutants, e.g., 8 colonies are picked up and streak plated. Then, a PCR reaction is set up with genetic material from each colony. A gel-electrophoresis is then run to investigate the size of the genetic material. This reveals whether the bacteria contain the desired plasmid.

# Plasmid purification of product from Gibson assembly

In this step, the plasmids from a successful Gibson assembly reaction were purified using the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit (Article no. 80101), from Cube Biotech. After purification, the DNA concentration was measured and the purified plasmids were then stored in the freezer (−20℃), ready for Golden Gate cloning.

For more details on how plasmid purification was performed, please see SOP 14.

# Golden Gate cloning to replace RFP with a dehalogenase

In this step, the placeholder RFP was replaced with one of the dehalogenases using Golden Gate cloning. One of the advantages of Golden Gate cloning is that the DNA cutting and ligation steps can be combined in a single reaction. The plasmid cannot re-assemble without a dehalogenase gene. This is because the overhangs created by the PaqCI in the backbone are not complementary.

For more details on Golden Gate cloning, including the program used in the PCR-machine, please see SOP 11.

# Transformation into NEB C3040I cells

In this step, the Golden Gate assembly is transformed into NEB® Stable Competent E. coli (High Efficiency) (Cat. No. C3040I), referred to as NEB C3040I. 2 µL of the Golden Gate mixture was added to the cells, which were then transformed by following a heat-shock protocol. Then, they were plated out on selection plates with 100 μg/mL ampicillin. NEB C3040I cells do not encode ampicillin resistance on their genome, but pET-51b does. Therefore, ampicillin was used as a negative selection marker.

For more details on the used heat-shock protocol, please see SOP 13.

# Colony PCR

Colony-PCR was used to verify whether the plasmid was assembled correctly after Golden Gate cloning. The dehalogenases are approximately the same length as the placeholder RFP. This means that, when amplifying the entire insertion piece, it was nearly impossible to see if the respective dehalogenase had successfully replaced RFP. Therefore, a new set of primers was designed. The forward primer bound to the dehalogenase gene but was unable to bind to RFP. The reverse primer bound to the plasmid backbone. Therefore, the DNA-product was only amplified if RFP had been successfully replaced by a dehalogenase.

Colony PCR was done on a total of 8 colonies per transformation. After gel electrophoresis, two of the colonies that contain the plasmid with the respective dehalogenase are saved as a freeze stock. For an overview of colony-PCR workflow, please see figure 11.

For a more detailed description of how colony-PCR was run, please see SOP 15.

![Colony-PCR](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/colony-pcr-1.svg)

**Fig. 11:** Overview of colony-PCR workflow. From a plate of successful mutants, e.g., 8 colonies are picked up and streak plated. Then, a PCR reaction is set up with genetic material from each colony. A gel-electrophoresis is then run to investigate the size of the genetic material. This reveals whether the bacteria contain the desired plasmid.

# Plasmid purification of product from Golden Gate cloning

Purification of the Golden Gate assembled plasmid was necessary at this time of the project. This is because NEB C3040I lacks a T7 promotor which is necessary for the expression of the dehalogenases and thereby the selection of the most efficient variant dehalogenases. After purification, the plasmid was transformed into the T7 Express Competent E. coli strain (High efficiency) (Cat. No. C2566I)

**An important note:** This Golden Gate cloning was performed before NEB C2566I was used. NEB C2566I could accept the direct product of Golden Gate cloning.

For more details on how plasmid purification was performed, please see SOP 14.

# Transformation of BBa\_K4868001 into NEB Express Competent Cells

In this step, the successful Golden Gate product was transformed into NEB C2566I, using a heat-shock protocol. The success of the transformation was tested by being plated out on selection plates with 100 μg/mL ampicillin and 0.4 mM IPTG. Ampicillin was used as a positive selection marker, and IPTG was used as a negative selection marker. This is because, if Golden Gate had failed, the cells would still express RFP. The NEB C2566I cells do not encode ampicillin resistance on their genome, whereas the plasmid does. Therefore, ampicillin was used as a negative selection marker for the transformation. IPTG was used as a negative selection marker for the Golden Gate cloning. This is because NEB C2566I contains a genomic T7 polymerase, which is IPTG-inducible. Had RFP not been replaced by a dehalogenase, cell cultures would therefore gain a red color in IPTG-rich media.

For more details on the heat-shock protocol, please see SOP 13.

# Riboswitch tests

With the plasmid successfully transformed into NEB C2566I, the functionality of the riboswitch, and thereby the proposed selection system was tested. The sensitivity of the riboswitch was tested by growing bacterial cultures in varying amounts of NaF. This was to prove that the fluorescence of the cells was correlated to the concentration of fluoride. The fluorescence of the cells was investigated in several ways; please read more about the different methods below, see figure 12.

![rib-test](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/riboswitch-test3.svg)

**Fig. 12:** Overview of riboswitch-tests performed on NEB C2566I. Firstly, day-cultures of ALE1 containing the plasmid of interest were set up in different NaF-concentrations. These were then investigated using fluorescence miscroscopy, in 96-well plates on a plate-reader and on agar-plates under a blue-light transilluminator.

# Fluorescence microscopy to test riboswitch sensitivity in NEB C2566I

In preparation for fluorescence microscopy, day-cultures with varying NaF concentrations were made and induced with 0.4 mM IPTG. The fluorescence of the cells was then analyzed under the microscope. This was useful for both investigating whether GFP-expression increased with the NaF-concentration and if there was any remaining RFP-induction after the Golden Gate cloning.

For more details on the experimental setup for fluorescence microscopy imaging, please visit SOP 17.

# Assessing the fluorescence of bacterial cultures using a plate reader

Aside from assessing cell fluorescence under the microscope, quantitative data was needed to confirm the observations from the fluorescence microscope. Thus, a 96-well plate with a range of NaF concentrations was prepared. Both OD600 and fluorescence was measured on a plate reader. The collected data confirmed that the fluorescence of cells containing the plasmid did increase with the NaF-concentration.

# Analyzing fluorescence of single bacterial colonies on agar plates

It was tested whether it was possible to see an increase in fluorescence on agar plates with increasing NaF concentrations. The fluorescence was measured with a blue light transilluminator. Although not a quantitative method, this approach might be useful to preselect the most fluorescent colonies when selecting the most improved enzymes.

# FACS

Even though each of the methods so far confirmed the activity of the riboswitch, none of them were ideal for the selection of the improved dehalogenases. Using FACS made it possible to collect valuable fluorescence data for a more detailed analysis of the bacterial cultures.

For a more detailed description of how FACS was conducted, please visit SOP 22.

# Error-prone PCR of dehalogenase genes

To optimize the dehalogenases, mutations were introduced in the genes encoding the dehalogenases, generating multiple variants. For this purpose, the JBS Error-Prone Kit (Cat. No. JBS PP-102) was used. This kit includes a Taq DNA-polymerase that lacks proofreading abilities, a special dNTP mix, and other components that contribute to a mutagenesis rate of 19%. By running the error-prone PCR-program on the dehalogenase genes before Golden Gate cloning, no mutations were introduced to the plasmid backbone. Therefore, [BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001) retains its functionality, except for the dehalogenase introduced with Golden Gate cloning. This means that any increase in fluorescence of cells containing a variant dehalogenase comes from an improved enzymatic activity and not from elsewhere.

# Golden Gate cloning with variant dehalogenases

In this step, the placeholder RFP is replaced with one of the variant dehalogenases, using Golden Gate cloning. One of the advantages of Golden Gate cloning is that the DNA digestion and ligation steps can be combined in a single reaction. The plasmid cannot re-assemble without a dehalogenase gene. This is because the overhangs created by the HindIII and SgrAI in the backbone are not complementary.

For more details on Golden Gate cloning, including the program used in the PCR machine, please see SOP 11.

# Transformation into competent expression strain

The Golden Gate product was transformed into NEB C2566I Express competent E. coli cells, by following a heat-shock protocol. Whether transformation was successful was tested by plating out the bacteria on selection plates with 100 μg/mL ampicillin, 0.4 mM IPTG, and 1.2 mM PFOA. Ampicillin was used as a positive selection marker, and IPTG was used as a negative selection marker. This is because, if Golden Gate had failed, the plasmid would still contain RFP. This means that any bacterial culture grown on IPTG would be red.

From previous experimentation, it was concluded that the PFOA concentration at its maximum solubility, 1.2 mM, is not enough to fully activate the riboswitch. Therefore, there was added 88 µL pr. cm2 of a 1.2 mM PFOA solution in water on top of the dried agar plates. Once dry, an even layer of PFOA was left on top of the plates.

For more details on the used heat-shock protocol, please see SOP 13.

# FACS selection

Because all colonies from the transformation were on plates, they were all washed off, resuspended, and diluted, so the FACS machine could measure fluorescence efficiently. The strategy was to sort the 0.1% most fluorescent single cells into wells on a 96-well plate. This was repeated for each dehalogenase. The 96-well plates were then incubated and streaked onto agar plates.

For a more detailed description on how FACS was conducted, please visit SOP 22.

# Purification and sequencing of plasmids containing variant dehalogenases

A total of 40 plasmids were purified and sent for sequencing. After receiving sequencing results, both the unmodified and variant dehalogenases were compared, to see which mutations had occurred.

For more details on how plasmid purification was performed, please see SOP 14.

# Plate reader selection

The variant dehalogenases were tested, by being plated onto agar plates with 1.2 mM PFOA, followed by overnight incubation at 37℃, allowing the bacteria to grow. Next, they were transferred to 27℃, being the optimal temperature of the enzymes. After tracking the fluorescence for 24 hours, the bacteria were washed off the plates, normalized, and had both OD600 and fluorescence measured. Then, wells that had the highest fluorescence compared to the OD600 were chosen for further experimentation.

# Protein purification

This process started with making overnight cultures of bacteria with the desired enzymes, and then large day cultures. Once they had reached the exponential phase, they were induced with 0.4 mM IPTG. Inducing the cells in their exponential phase gives the greatest protein yield. The cells were lysed using a French press and the proteins were then purified using the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit (Article No. 80101).

For more details on protein purification using the above-mentioned purification kit, please refer to SOP 25.

# Protein purification

**We intended to do the following but unfortunately we were unable to before the deadline:**

This process started with making overnight cultures of bacteria with the desired enzymes, and then large day cultures. Once they had reached the exponential phase, they were induced with 0.4 mM IPTG. Inducing the cells in their exponential phase gives the greatest protein yield. The cells were lysed using a French press and the proteins were then purified using the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit (Article No. 80101).

For more details on protein purification using the above-mentioned purification kit, please refer to SOP 25.

# SDS-page

To check the expression of the dehalogenases, an SDS page was performed. Both induced and uninduced samples were compared to confirm the presence of the respective dehalogenases.

For a thorough explanation of how to perform an SDS page using a 16% Tris-Glycine gel, please refer to SOP 21.

# SDS-page

**We intended to do the following but unfortunately we were unable to before the deadline:**

To check the expression of the dehalogenases, an SDS page was performed. Both induced and uninduced samples were compared to confirm the presence of the respective dehalogenases.

For a thorough explanation of how to perform an SDS page using a 16% Tris-Glycine gel, please refer to SOP 21.

# F-NMR

F-NMR was another approach that could be used to test the efficiency of the variant dehalogenases. This method also allowed the investigation of all the byproducts of the PFOA degradation. Samples were made so the enzymes had 71 hours, 59 hours, and 47 hours to degrade PFOA. By comparing the spectra from each sample, the efficiency of the enzyme could be determined.

For more details on protein F-NMR, please refer to SOP 29.

# F-NMR

**We intended to do the following but unfortunately we were unable to before the deadline:**

F-NMR was another approach that could be used to test the efficiency of the variant dehalogenases. This method also allowed the investigation of all the byproducts of the PFOA degradation. Samples were made so the enzymes had 71 hours, 59 hours, and 47 hours to degrade PFOA. By comparing the spectra from each sample, the efficiency of the enzyme could be determined.

For more details on protein F-NMR, please refer to SOP 29.

# Fluoride probe

ALE1 and ALE2 were developed to have more fluoride tolerance since the fluoride released from PFOA degradation will become toxic upon accumulating. The increased tolerance was achieved by Assisted Laboratory Evolution. The Escherichia coli Rosetta strain was selected for this experiment since it has a T7 promotor, which plays a crucial part in our selection system for improved enzymes. The basis of the system is that when the cells are induced with IPTG, the T7 polymerase will be expressed, which then will allow the expression of the respective dehalogenase encoded in our plasmid.

# Fluoride probe

**We intended to do the following but unfortunately we were unable to before the deadline:**

ALE1 and ALE2 were developed to have more fluoride tolerance since the fluoride released from PFOA degradation will become toxic upon accumulating. The increased tolerance was achieved by Assisted Laboratory Evolution. The Escherichia coli Rosetta strain was selected for this experiment since it has a T7 promotor, which plays a crucial part in our selection system for improved enzymes. The basis of the system is that when the cells are induced with IPTG, the T7 polymerase will be expressed, which then will allow the expression of the respective dehalogenase encoded in our plasmid.

# *E. Coli* Rosetta

ALE1 and ALE2 were developed to have more fluoride tolerance since the fluoride released from PFOA degradation will become toxic upon accumulating. The increased tolerance was achieved by Assisted Laboratory Evolution. The *Escherichia coli* Rosetta strain was selected for this experiment since it has a T7 promotor, which plays a crucial part in our selection system for improved enzymes. The basis of the system is that when the cells are induced with IPTG, the T7 polymerase will be expressed, which then will allow the expression of the respective dehalogenase encoded in our plasmid.

# ALE1

Gradually, the bacteria were exposed to increased NaF concentrations in LB-media. Additionally, they were exposed to UV light for a short time to induce mutations, thereby speeding up the adaption process. An illustration of the experimental workflow can be seen on figure 13.

For further details on how Adaptive Laboratory Evolution was used to increase the fluoride tolerance of E. coli Rosetta in liquid media, please visit SOP 31.

![ale1](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/ale1-96-well-plate.svg)

**Fig. 13:** Illustration of how the ALE1 experiments were set up. The ALE1 strain was developed in liquid media on 96-well plates, with a dilution row of NaF, and then exposed to UV-light to speed up the evolution process.

# Transformation of pET-51b containing BBa\_K4868001 into ALE1

Both the purified plasmid from Gibson assembly and Golden Gate cloning plasmid were transformed into the ALE1-strain using a heat-shock transformation.

For more details on the heat-shock protocol, please visit SOP 13.

# Riboswitch tests of ALE1

With the plasmid successfully transformed into ALE1, the functionality of the riboswitch, and thereby the selection system was tested. The sensitivity of the riboswitch was tested by growing the bacteria in different NaF concentrations. In theory, the activity of the riboswitch, and thereby fluorescence should therefore gradually increase. The activity of the riboswitch was tested in three different ways: with fluorescence microscopy, a plate reader, and with a blue light transilluminator. An overview of the methods can be seen in figure 14.

![rib-test](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/riboswitch-test3.svg)

**Fig. 14:** Overview of riboswitch-tests performed on ALE1. Firstly, day-cultures of ALE1 containing the plasmid of interest were set up in different NaF-concentrations. These were then investigated using fluorescence miscroscopy, in 96-well plates on a plate-reader and on agar-plates under a blue-light transilluminator.

# Microscopy

The experimental procedure was the same as described further up on this page. For comparison, this experiment was also run in NEB C3040I cells and the original Rosetta strain. Surprisingly, ALE1 showed much lower fluorescence intensity than NEB C3040I and Rosetta. It was theorized that the increased fluoride tolerance from ALE1 might come from optimized efflux pumps. Decreased fluorescence meant that it would be unfavorable to use the ALE1-strain to select for improved enzymes later.

For more information on the results from fluorescence microscopy, please visit our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

# Assessing the fluorescence of bacterial cultures using a plate reader

Aside from assessing cell fluorescence under the microscope, quantitative data was needed to confirm the observations from the fluorescence microscope. Thus, a 96-well plate with a range of NaF concentrations was prepared. Both OD600 and fluorescence was measured on a plate reader. However, the results were not what we had hoped for, and so, we had to consider what to do next.

For further details on the results from the plate-reader experiment, please visit our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

# Analyzing fluorescence of single bacterial colonies on agar plates

It was tested whether it was possible to see an increase in fluorescence on agar plates with increasing NaF concentrations. The fluorescence was measured with a blue light transilluminator. Although not a quantitative method, this approach might be useful to preselect the most fluorescent colonies when selecting the most improved enzymes.

# ALE2

This strain was developed, by plating them on agar plates and exposing them to gradually increasing NaF-concentrations over time. It was assumed that this would make the bacteria develop a higher tolerance to fluoride. Additionally, the plates with the bacteria were exposed to UV light for 15 seconds to induce mutations, thus accelerating the evolution process. An illustration of the experimental setup can be seen on figure 15.

Performing the experiment on agar plates gave the advantage of more easily tailoring the NaF-concentration to the tolerance of the bacteria each day, compared to the used dilution row. The disadvantage was that plates had to be cast each day, allowing room for uncertainty of measurement.

For further details on how Adaptive Laboratory Evolution was used to increase the fluoride tolerance of *E. coli* Rosetta in solid media, please visit SOP 32.

![ale2](https://static.igem.wiki/teams/4868/wiki/illustrations-images/design/ale-2-1.svg)

**Fig. 15:** Illustration of how the ALE2 experiments were set up. The ALE2 strain was developed in solid media on agar plates, with a dilution row of NaF, and then exposed to UV-light to speed up the evolution process.

# Transformation of BBa\_K4868001 into ALE2

Both the purified plasmid from Gibson assembly and Golden Gate cloning plasmid were transformed into the ALE2-strain using a heat-shock transformation.

For more details on the heat-shock protocol, please visit SOP 13.

# Riboswitch tests of ALE2

Since ALE2 was developed separately from ALE1, it was theorized that the riboswitch tests would yield different results. The sensitivity of the riboswitch in ALE2 was tested in the same ways as ALE1; under a fluorescence microscope, with a plate reader, and with a blue light transilluminator.

An overview of the methods can be seen in figure 16.

![rib-test](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/riboswitch-test3.svg)

**Fig. 16:** Overview of riboswitch-tests performed on ALE2. Firstly, day-cultures of ALE1 containing the plasmid of interest were set up in different NaF-concentrations. These were then investigated using fluorescence miscroscopy, in 96-well plates on a plate-reader and on agar-plates under a blue-light transilluminator.

# Microscopy

To test the fluorescence system, we made day-cultures with increasing concentrations of NaF, ranging from 0 to 200 mM NaF. For comparison, we ran the same experiment parallel in NEB stable competent cells. The results of this experiment showed promising results, compared to ALE1. For further details, please visit our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page. However, the strain could not accept the product of Golden Gate cloning. Therefore, it was concluded that ALE2 was unfit for continued use in our experiments.

For more information on the results from fluorescence microscopy, please visit our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

# Assessing the fluorescence of bacterial cultures using a plate reader

Aside from assessing cell fluorescence under the microscope, quantitative data was needed to confirm the observations from the fluorescence microscope. Thus, a 96-well plate with a range of NaF concentrations was prepared. Both OD600 and fluorescence was measured on a plate reader. However, the results were not what we had hoped for, and so, we had to consider what to do next.

For further details on the results from the plate-reader experiment, please visit our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

## The Wall

It was official. We hit a wall. The working theory was that the increased fluoride tolerance came from optimized efflux pumps. This meant that both strains could maintain a very low intracellular concentration of fluoride. To verify this proposal, both ALE1 and ALE2 were sent to whole genome sequencing. Whole genome sequencing would also reveal why ALE2, but not ALE1 showed fluorescence. In any case, both ALE strains were unfit for the selection of optimized enzymes. So, the tale of ALE ended here. Not all hope was lost, though! Other strains still responded well to fluoride under the fluorescence microscope.

For further information, please visit our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

Unfortunately, the whole-genome sequencing results of ALE1 and ALE2 arrived just before the wiki-freeze, leaving too little time for analysis. The analysis will be ready for The Grand Jamboree, so stay tuned!

![wall](https://static.igem.wiki/teams/4868/wiki/illustrations-images/experiments/mur.svg)

# Standard Operating Procedures (SOP)

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4868/wiki/design-page/sop-den-der-skal-p-wiki-1.pdf) instead.

## Results

[![next-page-button](https://static.igem.wiki/teams/4868/wiki/wiki-images/next-page.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

## References

1. Engler, C., & Marillonnet, S. (2014). Golden Gate Cloning. In S. Valla & R. Lale (Eds.), DNA Cloning and Assembly Methods (pp. 119-131). Humana Press. https://doi.org/10.1007/978-1-62703-764-8\_9
2. Bioscience, J. JBS Error-Prone Kit - Random Mutagenesis by Error-Prone PCR https://www.jenabioscience.com/molecular-biology/cloning-and-mutagenesis/random-mutagenesis-kits/pp-102-jbs-error-prone-kit
3. Biotech, C. HisCube Ni-INDIGO - His-tag Protein Purification MINI Kit. https://cube-biotech.com/media/97/e2/2f/1666611404/80101%20INDIGO-Ni%20%20MINI%20Kit.pdf
4. Southern University of Denmark, NMR-Spektroskopi. https://www.sdu.dk/da/om\_sdu/institutter\_centre/fysik\_kemi\_og\_farmaci/samarbejde/bestil-analyser/nmr/teori

{% endblock %}

{% extends "layout-new.html" %}
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# Results

* [Testing inducibility](#testing-inducibility)
* [GFP Fluorescence Distribution](#fluorescence-distribution)
* [Evaluating performance of selected DeHa versions](#selected-deha-versions)
* [Insertion and sequencing of the mutant DeHa variants](#mutant-deha-variants)
* [Protein Purification](#protein-purification)
* [Supplementary Results](#supplementary-results)

# Overview

We started the development of our VitroZymes in the laboratory by testing the sensitivity of the riboswitch ([BBa\_K4868000](http://parts.igem.org/Part%3ABBa_K4868000)) in our live-cell system, which has been undocumented so far. This experiment laid the foundation for the utilization of the selection system encoded in our insertion piece called RGFr ([BBa\_K4868001](http://parts.igem.org/Part%3ABBa_K4868001)). Further testing was required to confirm the induction of the respective dehalogenases in IPTG-rich media. Additionally, the enzymatic activity of two of the original enzymes, DeHa 4 ([BBa\_K4868998](http://parts.igem.org/Part%3ABBa_K4868998)) and DeHa 5 ([BBa\_K4868999](http://parts.igem.org/Part%3ABBa_K4868999)) was investigated using F-NMR. Lastly, we planned to compare the enzyme kinetics of the original dehalogenases, DeHa 1, 2, 4, and 5 to the most fluorescent dehalogenase variants from a mutant library created with error-prone PCR. Unfortunately, this last experiment was not performed due to hardware delivery issues.

# Testing the inducibility of the fluoride sensitive riboswitch of RGFr

Before it is possible to use the riboswitch efficiently it is necessary to test if it works and how much fluoride is required to measure a difference in the sfGFP expression.

Our plasmid consists of RGFr where there is **a constitutive active promotor followed by the riboswitch and a superfolder Green fluorescent protein (sfGFP).** The riboswitch controls the translation of sfGFP and is first activated when fluoride ions are present in the cell1. This means that the greater the amount of fluoride present in the cell, the more sfGFP will be translated. Hence, when our Dehalogenases degrade PFOA the fluoride concentration will increase, resulting in greater amounts of expressed sfGFP. **Therefore, more efficient variants of the DeHas will generate a higher amount of fluoride which will lead to an increased expression of GFP, which enables efficient selection of our optimized dehalogenase enzymes.**

In the article the riboswitch has been tested in a **cell-free system**, and it was found that concentrations as low as **0.1 mM1 affect the activity of the riboswitch.** See Figure 1 on the Design page for a more graphic illustration.

However, live-cell systems, such as the one used for the selection of the most efficient enzymes, differ in how they work, since there are different pathways and proteins in a bacterium that cannot be replicated in a cell-free system. Fluorescence microscopy was done for qualitative evaluation of the fluoride induced sfGFP, for NEB T7 (NEB #C2566I) containing our plasmid which is grown in varying fluoride concentrations (Fig. 1).

The NEB T7 (NEB #C2566I) strain showed a gradual increase in GFP intensity in increased NaF concentrations. **Low levels of sfGFP could also be observed in the presence of 0 mM NaF, indicating that the riboswitch has leakage.** sfGFP distribution throughout the population seemed to be not entirely uniform.

![Fmicroscope-NEBT7-fig](https://static.igem.wiki/teams/4868/wiki/results/nebt7-c25561.jpg)

**Fig. 1:**Fluorescence microscopy images of NEB T7 cells (NEB #C2566I) carrying the RGFr containing plasmid with fluoride inducible sfGFP expression at 0, 47, 84, and 112 mM NaF. The image shows the increase in sfGFP expression in NEB cells was increased with the NaF concentration.

## Quantitative assessment of the riboswitch induction via FACS and plate reader experiments

FACS and plate reader were used to assess the fluoride inducibility of the riboswitch controlled sfGFP in the NEB T7 *E. Coli* strain (NEB #C2566I).

In the FACS and plate reader experiment the results show that the riboswitch needs around 50 mM NaF in most of the plasmids to register a change in sfGFP expression (Fig 2). This indicates that the riboswitch sensitivity is low, which could cause problems when testing for PFOA degradation. This is because PFOA has a solubility of 1.2 mM in water which would result in a fluoride concentration of 16.8 mM PFOA which might be too low to observe a fluorescence response. As a solution for this we grew them on agar plates covered with a PFOA layer.

![F-plate-fig](https://static.igem.wiki/teams/4868/wiki/results/fig2-riboswitchplate.jpg)
![F-FACS-fig](https://static.igem.wiki/teams/4868/wiki/results/fig3-facsriboswitch.jpg)

**Fig. 2:** *Left:* Plate reader experiment, which measured fluorescence with various gene insertions (DeHa1, 2, 4 and 5) into the insertion site of RGFr. NEB WT shows the fluorescence of the wild type NEB T7 (NEB #C2566I) strain without any plasmid. sfGFP fluorescence is normalized to the optical density.
*Right:* Flow-cytometry measurements of the same cultures. The 99 percentile (top 1% fluorescent cells) of sfGFP fluorescence (FITC-A) is shown for each strain.

# GFP Fluorescence Distribution of Cells Containing the Original Dehalogenases

**It was necessary to access the effectivity of the original dehalogenases and therefore an experiment was set up using FACS and violin plots (Fig. 3).** This experiment determined the distribution of fluorescence from sfGFP in samples with the different DeHAs, a sample containing RFP at the insertion site. It can be seen from the figure below that the riboswitch is inducible with fluoride in all the plasmids harboring DeHas (Fig. 4-7). Notably, it can be observed that there are often fluorescent and non-fluorescent populations within the same sample. No fluorescence induction could be observed in wild type (NEB #C2566I) strain (Fig. 8). Notably a big fluorescence population is observable at 0 mM NaF. This indicates a leakage of the riboswitch.

![fig3-violin](https://static.igem.wiki/teams/4868/wiki/results/violin-plot-1.png)

**Fig. 3:** Violin plot of GFP fluorescence from NEB T7 (NEB #C2566I) containing RGFr. It can be seen that the higher the concentration of NaF the more fluorescence can be detected for RGFr (BBa\_K4868001).

![fig4-violin](https://static.igem.wiki/teams/4868/wiki/results/violin-plot-2.jpg)

**Fig. 4:**Violin plot of GFP fluorescence from NEB T7 (NEB #C2566I) containing RGFr with DeHa 1 (BBa\_K4868996).

![fig5-violin](https://static.igem.wiki/teams/4868/wiki/results/violin-plot3.png)

**Fig. 5:** Violin plot of GFP fluorescence from NEB T7 (NEB #C2566I) containing RFGr with DeHa 2.

![fig6-violin](https://static.igem.wiki/teams/4868/wiki/results/violin-plot-4.jpg)

**Fig. 6:** Violin plot of GFP fluorescence from NEB T7 (NEB #C2566I) containing RFGr with DeHa 4.

![fig7-violin](https://static.igem.wiki/teams/4868/wiki/results/violin-plot-5.jpg)

**Fig. 7:** Violin plot of GFP fluorescence from NEB T7 (NEB #C2566I) containing RGFr and DeHa 5.

![fig8-violin-control](https://static.igem.wiki/teams/4868/wiki/results/fig-xx-violin.jpg)

**Fig. 8:** Violin plot of GFP fluorescence from NEB T7 cells wildtype (NEB #C2566I). Roughly the same level of fluorescence can be seen in all the samples at all the tested concentrations of NaF.

## Selecting for efficient error-prone DeHas via FACS

To obtain a variety of different DeHas (DeHa1, 2, 4, and 5 respectively) an error-prone PCR was performed. Golden Gate was performed to insert the error-prone DeHas into the RGFr carrying plasmid, resulting in a diverse mix of plasmids with slightly different versions of the respective DeHa. Subsequently this DeHa mutant libraries were transformed into NEB T7 (NEB #C2566I) and plated out on LB-media plates with an antibiotic for selection and a PFOA layer (88 µL/cm2 of evaporated 1.2 mM PFOA solution). Therefore, colonies with more efficient DeHa versions would result in higher fluorescent cells.

The plates were incubated overnight at 37°C for optimal growth of the bacteria and were then moved to 25°C, since it was estimated that **the enzymes have a higher efficiency at that temperature.**

The selection of our highly efficient mutated DeHas was performed using FACS. The samples were prepared by resuspending and pooling all colonies of a transformation plate, mixing them thoroughly. This was performed for each DeHa (1, 2, 4, and 5) respectively. The single **top 0.1%** fluorescent cells were sorted at 2000 events/second into 96-well plates. **40 variants from all DeHas** that showed subsequently the highest fluorescence on the 96-well plate in the plate reader were sent for sequencing.

**Furthermore, the fluorescence profile of the original DeHa populations (also grown on PFOA plates) were measured and compared to the fluorescent profile of the mutant populations (Fig. 9)**  The recorded data were used to compare the fluorescence distribution of each original DeHas compared to the corresponding mutated dehalogenase pool (Fig. 9).

![fig9-violin](https://static.igem.wiki/teams/4868/wiki/results/error-prone-violin-plot1.jpg)

**Fig. 9:** Violin plots that show the distribution of fluorescence of cells containing each original dehalogenase compared to its mutated counterparts. A is DeHa 1, B is DeHa 2, C is DeHa 4 and 5 is DeHa 5. By comparing the original enzymes (yellow plot) with the mutated ones (blue plot), it can be seen that the mutated DeHa 2 and 5 fluorescence are more than the original variant, For DeHa 4 and 1 there is no significant difference, and DeHa 1 have gotten worse.

When looking at Figure 9, a clear difference can be observed between the original DeHa and the mutated DeHa pool of DeHa 2 and DeHa 5. **Based on the plots of DeHa 2 and DeHa 5 it indicates that the original DeHa 2 and DeHa 5 are not very efficient at degrading PFOA compared to the mutated counterparts, under the same conditions.** However, DeHa 1 and DeHa 4 show approximately the same level of sfGFP as their mutated counterparts. Considering that there were thousands of variants generated per DeHa to begin with and only a small fraction of the pooled variants were analyzed, there is a certain likelihood that no beneficail mutations were present in this pool of mutants.

## Evaluating performance of selected DeHa versions

The selected 40 variants were tested in a cell system, where each mutation variant, and the original DeHas, were plated out on agar plates with a concentrated PFOA layer in the same manner as for the plates for the FACS experiment. Fluorescence of the colonies was monitored with exposure to blue light in the GelDoc (BIO-RAD 10000125685) to determine which of the plates had the most fluorescent colonies. For further details on how this experiment was conducted, please visit our Experimental Design page. A side-by-side comparison in fluorescence of the mutated variants and original DeHas can be seen in figure 10.

![fig10-F-bargraph](https://static.igem.wiki/teams/4868/wiki/results/mutated-deha-f-bar-graph.jpg)

**Fig. 10:** Fluorescence comparison of the mutated DeHa variants and original DeHas. Original DeHas are marked in red, DeHa 1 is marked in blue, DeHa 2 is marked in green, DeHa 4 is marked in purple and Deha 5 is marked in yellow. The black error bars indicate the variation in fluorescence measured between the triplicates of each variant.

The pattern that can be seen in Figure 10 matches the previously obtained FACS data seen in Figure 9. **Both the mutated DeHa 2 and DeHa 5 variants show an overall increase in fluorescence compared to the original DeHa 2 and DeHa 5**, whereas the fluorescence of the mutated DeHa 1 and DeHa 4 variants does not show an overall increase in fluorescence. **Based on Figure 10 it can be concluded that most of the DeHa 2 and DeHa 5 mutant variants express an increased amount of sfGFP compared to the original DeHa 2 and DeHa 5.** Since the expression of sfGFP comes from an increased amount of fluoride, which activates the riboswitch, it can be assumed that the increase in fluorescence is due to higher efficiency of the mutated DeHas. Whether the enzymes show the same increase in efficiency in vitro making them applicable still needs further investigation.

## Insertion and sequencing of the mutant DeHa variants

Sequencing of the selected mutant variants confirmed the insertion of 7 DeHa 1 variants, 9 DeHa 2 variants, 7 DeHa 4 variants, and 7 DeHa 5 variants. **When taking a closer look at the translations of the mutant variants it could be seen that a lot of the mutant variants had mutations that led to one or several stop-codons in the amino acid sequence.** The amino acid sequences of the amino acids were aligned to see if there were any locations where several variants have mutations to determine sites of the enzymes that most likely are essential for the activity of the enzyme. Figure 11 shows an alignment of the DeHa 1 variants, where the stop codon in DeHa1\_var1 and the regions with mutations are highlighted.

![fig11-alignmentDeHa1](https://static.igem.wiki/teams/4868/wiki/results/alignment-deha1.jpg)

**Fig. 11:** Alignment of DeHa1 variant amino acid sequences compared to the original DeHa1. The blue colors indicate the identity percentage, and the red highlights mark the stop codon and positions where multiple variants have mutations.

The same alignment was done for the DeHa 2 variants (Figure 12). There were however no DeHa 2 variants that didn’t have a stop codon. Furthermore, DeHa2\_var3 and DeHa2\_var4 had frameshift mutations, which is why they were excluded from the alignment. **The frameshift mutations might however still have had a gain-of-function impact on the enzyme structure, since the fluorescence experiment showed high fluorescence in DeHa2\_var4.** All of the stop codons and positions where multiple variants have mutations are highlighted in Fig. 12

![fig12-alignmentDeHa2](https://static.igem.wiki/teams/4868/wiki/results/alignment-deha2.jpg)

**Fig. 12:** Alignment of DeHa2 variant amino acid sequences compared to the original DeHa 2. The blue colors indicate the identity percentage, and the red highlights mark the stop codons and positions where multiple variants have mutations.

In the alignment of DeHa 4 and its variants (Figure 13), multiple variants had frameshift mutations and those were excluded from the alignment. Since there also was not seen an increase in fluorescence of the mutated DeHa 4 variants compared to the original DeHa 4 the other DeHas were prioritized for further analysis.

![fig13-alignmentDeHa4](https://static.igem.wiki/teams/4868/wiki/results/aligment-deha4.jpg)

**Fig. 13:** Alignment of DeHa4 variant amino acid sequences compared to the original DeHa 4. The blue colors indicate the identity percentage, and the red highlight marks the stop codon.

Two of the DeHa 5 mutant variants had frameshift mutations which is why they were excluded from the alignment (figure 14). Furthermore, the overall mutation rate was lower compared to the other DeHas. **The DeHa 5 mutant variants did however show the biggest increase in fluorescence compared to the original DeHa 5.**

![fig14-alignmentDeHa5](https://static.igem.wiki/teams/4868/wiki/results/alignment-deha5.jpg)

**Fig. 14:** Alignment of DeHa5 variant amino acid sequences compared to the original DeHa5. The blue colors indicate the identity percentage.

Though the sequencing results show many variants containing one or more stop codons it does not necessarily mean that the mutated variants DeHas will not be fully transcribed or functional as enzymes. For example, it has been reported that bacteria (such as *E. coli*) have varying UGA readthrough, which means that **bacteria can in certain situations transcribe past a stop codon(2).**

Other studies supported varying efficiencies for *E. coli* to terminate transcription. Furthermore, another study suggested that the base pair following the stop codon dictates whether transcription halts or proceeds3. Other studies suggest that the stop codon is nothing more than an indication of where to terminate the signal and there are many other factors needed for transcription termination4.

There is also a possibility that the enzymes could be transcribed until the stop codon, and folded, while maintaining their enzymatic pocket and thereby still be functional enzymes. Therefore, the stop codons are not to be seen as determining factors for the possible further investigations of the mutated variants enzymes. **Additionally, the sequencing results revealed the cells showing the most fluorescent when running them in FACS, had in fact mutations forming stop codons in their sequence.**

Additionally, docking was used to simulate the binding of PFOA to the mutated DeHa variants. See more on this [link](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

## Protein purification

For testing the DeHas in a cell-free system they had to be purified. First, there had to be proven DeHa expression. This was done by purifying them using a His-tag purification kit (Cube Biotech 80101) and then running the purified proteins on an SDS page (Fig. 15). DeHa 1 and DeHa 5 were run on a gel together and DeHa 2 and 4 on another gel, as seen on figures 15. As expected, the bands for Deha 4 and Deha 5 are visible at ~34 kDa and ~25 kDa respectively. Conversely, for Deha 1 and DeHa 2, there were no clear bands at the expected size of 26 kDa. The expected protein sizes can be seen in table 1. Likely the enzymes were nonetheless expressed, but the purification protocol remains to be optimized for these enzymes, as they neigher could be found in the flow-through or wash fractions of their respective purification (data not published).

If there was more time at disposal, it would have been possible to optimize the purification process using different temperatures and different elution solutions for instance.

**Table 1)** Overview of molecular weight of RFP and dehalogenases 1, 2, 4, and 5, both with and without a 6x His-tag.

|  | Mol. weight (kDa) | Mol. weight w/ 6x His-tag (kDa) |
| RFP | 25.30 | - |
| DeHa 1 | 25.69 | 26.53 |
| DeHa 2 | 25.51 | 26.35 |
| DeHa 4 | 33.6 | 34.44 |
| DeHa 5 | 24.14 | 24.98 |

![fig15-deha-1-5-2-gel](https://static.igem.wiki/teams/4868/wiki/results/fig-14-deha-1-5-2-gel.jpg)
![fig15-deha-2-2-4-gel](https://static.igem.wiki/teams/4868/wiki/results/fig-14-deha-2-2-4-gel.jpg)

**Fig. 15:** 2 SDS-page results from purified samples of original DeHa 1, 5 and 2 (left) and DeHa 2 and 4 (right). For size reference, the protein ladder PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Cat. No. 26619). Each dehalogenase was run in duplicates of 5, corresponding to each elution step of the protein purification. This was done to investigate which elution step yielded the most protein. On the left image, no bands show up for DeHa 1, nor for DeHa 2. DeHa 5 yields protein in each elution step. On the right image, DeHa 2 shows several bands, but not around 25 kDa, as expected. Duplicates 3-5 of DeHa 4 shows strong bands in the range between 25 kDa and 35 kDa.

## F-NMR

**Fluorine-19 nuclear magnetic resonance spectroscopy (19F-NMR) was used to prove the enzymes did break down PFOA.** For this purpose, one sample was taken after 80 hours, 68 hours, and 44 hours. 19F-NMR-spectra were obtained. The spectra can be seen below, in figure 16:

![fig16-FNMRspectra](https://static.igem.wiki/teams/4868/wiki/results/fig-15-fnmr-spectra.jpg)

**Fig. 16:** 97F-NMR spectra for solutions with PFOA and DeHa 4 and DeHa 5 after 80h, 68hr, and 44 hr. The spectra show no difference for the chemical shift or integral height. This indicates the enzymes did not work.

Unfortunately, there were no differences in the spectra when they were compared to the samples with PFOA. This indicated that the enzymes did not work on PFOA and therefore no C-F bonds were broken. This could have been due to the low concentration of enzyme (5 µg/mL), or the enzyme unfolded before they were able to interact with PFOA. One explanation could be that the enzymes have a degradation rate bigger than 80 hours. Additionally, temperature optimization could be investigated. The protocol for the used kit for protein purification could likewise be adjusted.

## Model

[![next-page-button](https://static.igem.wiki/teams/4868/wiki/wiki-images/next-page.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

# Supplementary results

## Adaptive Laboratory Evolution

*If you are interested in the Whole Genome Sequencing results for the ALE1 and ALE2 strains, please contact: sofielmoebes@gmail.com*

Prior to the plasmid assembly it was hypothesized that it would benefit the project if the bacteria could tolerate an increased amount of fluoride, as fluoride ions are released during the enzymatic degradation of PFOA. So, the first idea of the project was that the bacteria could be integrated into a closed system that could work as a live PFAS degradation filter. The aim was to enable the bacteria to tolerate a NaF tolerance of 200 mM.

Therefore, a side project was initiated aimed to create an *E. coli* with increased fluoride tolerance. However, the project and the corresponding results did not meet the criteria, and the side project was discontinued.

We aimed to increase this tolerance to 200 mM NaF using adaptive laboratory evolution (ALE). In the first ALE experiment, the bacteria were cultivated in a 96-well plate containing LB media with different NaF concentrations. Over a 4-week period, we inoculated a new 96-well plate containing a NaF dilution row with an aliquot of the well from the day before. We will refer from here on to the strain obtained through this ALE as ALE1. We attempted another ALE-experiment in which we grew the bacteria on LB-agar plates containing NaF. Each day, we streak-plated colonies onto fresh plates a higher NaF concentration than the day before. We refer to the mutant strain obtained from this experiment as ALE2 from here onwards. For an overview of the experimental setup, please refer to figure 9S.

![ale-experiment-set-up](https://static.igem.wiki/teams/4868/wiki/results/ale-experiments-set-up.png)

**Fig. 9S:** Figure showing the experimental setup for ALE1 and ALE2. ALE1 was created using liguid culture while ALE2 was created using solid culture. Created with biorender.com

To conclude the experiment, the growth of both ALE1 and ALE2 on different concentrations of NaF, ranging from 0 mM to 400 mM, was compared to that of the original Rosetta (Fig. 10S). The three different strains were plated out in a 1:10 serial dilution from a factor 1 to a factor of 10-7, on LB agar plates containing 0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, and 400 mM NaF. Overall, there are no remarkable differences in bacterial growth between the strains on plates with low (0 mM and 50 mM) NaF. On plates which contain 200 mM NaF a distinct difference in growth could be observed between the two strains. ALE1 does not show any clear difference in growth to the original Rosetta strain, with both only showing growth down to a dilution of 10-2 at a concentration of 200 mM. ALE2, conversely consistently grew a clear lawn down to a dilution of 10-3 at a concentration of 200 mM.

![10s-left](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/rosetta-vs-ale1-petri-9.png)
![10s-right](https://static.igem.wiki/teams/4868/wiki/illustrations-images/wet-lab-resultater/rosetta-ale-2-9-petri.png)

**Fig. 10S:**Final comparison experiment of growth of ALE1 & Rosetta (left), and ALE2 & Rosetta (right). Both ALE1 and Rosetta shows growth down to a culture dilution of 10-2
at a concentration of 200 mM. ALE2, conversely consistently grew a clear lawn down to a concentration of 10-3 at a concentration of 200 mM.

At concentrations above 350mM NaF, none of the cells grew.

At conclusion the ALE strains were not suitable for the purpose of this specific study. They, however, exhibit significant potential in studies that rely on the tolerance of *E. coli* to fluoride.

From the results above, it can be concluded that ALE2 does have increased fluoride tolerance, compared to the original Rosetta strain. Unfortunately, fluorescence microscopy images show that ALE1 and ALE2 are unsuitable for selection of optimized dehalogenases. Please see figure 11S for comparison of fluorescence of ALE1, NEB and original Rosetta.

![F-microscope](https://static.igem.wiki/teams/4868/wiki/results/11s-f-microscope.png)

**Fig. 11S:** Fluorescence microscopy images of ALE1 (with increased NaF-tolerance), NEB C3040I and original Rosetta, at 0, 50, 100 and 150mM NaF. The image shows that the GFP-expression of both NEB C3040I and Rosetta cells increases with the NaF-concentration, whereas the GFP-expression in ALE1 does not. A morphology change in NEB C3040I cells, especially, can be observed at 150mM NaF, indicating cellular stress.

## References

1. 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 Synth Biol, 9(1), 10-18. https://doi.org/10.1021/acssynbio.9b00347
2. Fan, Y., Evans, C. R., Barber, K. W., Banerjee, K., Weiss, K. J., Margolin, W., Igoshin, O. A., Rinehart, J., & Ling, J. (2017). Heterogeneity of Stop Codon Readthrough in Single Bacterial Cells and Implications for Population Fitness. Mol Cell, 67(5), 826-836.e825. https://doi.org/10.1016/j.molcel.2017.07.010
3. Poole, E. S., Brown, C. M., & Tate, W. P. (1995). The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli. Embo j, 14(1), 151-158. https://doi.org/10.1002/j.1460-2075.1995.tb06985.x
4. Tate, W. P., Poole, E. S., Dalphin, M. E., Major, L. L., Crawford, D. J., & Mannering, S. A. (1996). The translational stop signal: codon with a context, or extended factor recognition element? Biochimie, 78(11-12), 945-952. https://doi.org/10.1016/s0300-9084(97)86716-8

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