iGEM 2023 — Team dtu-denmark


{% extends "layout.html" %}
{% block page\_content %}

# Engineering

Photo: DTU

![]()

##### Menu

* [> Validation of TMS](#validation-of-tms-system)
* [> Design of new TMS](#design-of-new-tms-structures)
* [> Design of PFOA TMS](#design-of-pfoa-tms)
* [> Design of cell-free system](#design-of-a-cell-free-system)
* [> References](#references)

The engineering cycle is the cornerstone of innovation and problem-solving in the field of engineering. It is a fundamental approach to the design, execution, and learning of a complex challenge through a systematic and structured framework, which can be used to develop new products and create innovative solutions for a wide range of problems. This iterative process guides you through defined stages to refine and optimize the end-result, ensuring that the creation meets the standards of functionality, safety and efficiency. In short, the engineering cycle is a roadmap that informs but also inspires the creativity and problem-solving of engineers and designers.

This innovative process can be divided into two sections: research-based ideation and the Design-Build-Test-Learn cycle. The first part consists of research, including problem identification and definition of the requirements, followed by imagining possible solutions and establishment of an initial design. The second part is a cyclic, iterative process composed of the four stages in the name: designing, building, testing, and learning. The purpose is to repeat the cycle as needed with the goal of improving the experiment and increasing the precision and quality of the results.

![Innovation process](https://static.igem.wiki/teams/4811/wiki/images/engeneering-process.svg)

Figure 1: The engineering cycle.

Through our project, we have delved into each stage of this cycle and here we shed light on how we have used the methodology, tools and considerations it provides to drive our idea forward. Our work within this cycle can be divided in three sections, namely the validation of the TMS system, the design of new variations of this system with alternative aptamers, and the application of TMSs in the PFAS problem with the use of the PFOA aptamer. For each section, we have aimed at running through multiple iterations of the cycle after learning from our experiences.

## Validation of TMS system

### Iteration 1

*Design*

After deciding on constructing a biosensor based on a tRNA-Mimicking Structure (TMS), we set the goal to first validate the concept using the GFP aptamer as described by Paul *et al.* (2020). As explained in the [Design](https://2023.igem.wiki/dtu-denmark/design) section, the TMS system is based on two plasmids: a high-copy number that contains the TMS, [BBa\_K4811017](http://parts.igem.org/Part%3ABBa_K4811017), and a low-copy number plasmids that contains both GFP and mCherry, [BBa\_K4811005](http://parts.igem.org/Part%3ABBa_K4811005). The expression of the TMS is induced by IPTG and the expression of mCherry by L-arabinose. The expression of GFP has the Ptet promoter, which is induced by the antibiotics family of tetracycline.

*Build & Test*

We first bought the construct from IDT to test the induction of fluorescence, which was transformed into *E. coli* BL21(DE3). The results showed a successful induction of mCherry using arabinose.

We USER cloned the [BBa\_K4811004](http://parts.igem.org/Part%3ABBa_K4811004) and [BBa\_K4811003](http://parts.igem.org/Part%3ABBa_K4811003) from IDT into our pACYC184 backbone, creating the composite part [BBa\_K4811005](http://parts.igem.org/Part%3ABBa_K4811005). We also USER cloned [BBa\_K4811018](http://parts.igem.org/Part%3ABBa_K4811018) into pUC19 backbone. To check that the correct inserts were there, we sequenced and digested the plasmids, which indicated a successful cloning.

Co-transformation of [BBa\_K4811005](http://parts.igem.org/Part%3ABBa_K4811005) and [BBa\_K4811018](http://parts.igem.org/Part%3ABBa_K4811018) resulted in a reporter system, which was tested on varying levels of L-arabinose and IPTG. The results showed a successful induction of mCherry using arabinose, as well as repression of mCherry by IPTG, which expresses our TMS, the results of which can be seen below:

![TMS GFP 1 ](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/gfp-tms-varying-iptg.png)

Figure 2: mCherry fluorescence normalized to OD, 561 nm excitation/617 nm emission, as a function of IPTG concentration. All measurements were with 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with no inducers present (uninduced).

![TMS GFP 2](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/gfp-tms-varying-arabinose.png)

Figure 3: mCherry fluorescence normalized to OD, 561 nm excitation/617 nm emission, as a function of L-arabinose concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG (IPTG), one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced).

Furthermore, a Minimal Inhibitory Concentration (MIC) test was performed to determine the concentration of tetracycline that could be used without killing the cells. After a successful double transformation into *E. coli* BL21(DE3) and induction, however, there were no results and a low OD of the cells, indicating that the cells had died from the tetracycline.

*Learn*

From this introductory step, the results showed that 1 mM IPTG and 0.1 % w/v L-arabinose were sufficient conditions for simultaneous expression of mCherry and the TMS. We also learned that non-modified tetracycline is too potent for cells to survive with no expression of mCherry or the TMS. It is known that tetracycline inhibits the P site of the ribosome to hinder protein synthesis, which could explain why the cells could survive at certain low concentrations but no fluorescence could be measured.

### Iteration 2

*Design*

After determining that tetracycline was too potent to use, we looked into modified versions of tetracycline that are less potent and thus could be used instead. We decided to use anhydrotetracycline (aTc), since it seemed to work in the published references (Paul *et al.*, 2020).

*Build & Test*

The induction experiment was performed with the same setup as before with aTc instead of tetracycline. The OD showed an increase in growth and the measurements of GFP fluorescence increased when the aTc concentration increased. However, mCherry fluorescence did not change when aTc concentration increased.

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/gfp-tms-gfp-varying-atc.png)

Figure 4: GFP fluorescence normalized relative to OD, 488 nm excitation/545 nm emission, as a function of anhydrotetracycline (aTc) concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG (IPTG), one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced).

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/gfp-tms-mcherry-varying-atc.png)

Figure 5: mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of anhydrotetracycline (aTc) concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG (IPTG), one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced).

*Learn*

aTc was a good choice for induction of GFP and was not potent enough to kill or inhibit growth of *E. coli* BL21(DE3), with 0.5 μM aTc having the highest fluorescence.

When looking at the corresponding mCherry fluorescence, there is no significant difference between varying amounts of aTc. As such, we were not able to recreate the results of Paul *et al.* (2020), as GFP led to an increase in mCherry fluorescence in the paper. However, the control with L-arabinose was not significantly different from the experiment with 0 μM aTc. This leads us to believe that the TMSs ability to inhibit mCherry might have been compromised in our setup. We found this interesting, as our sequences and setup were designed to match the paper as closely as possible, but did not succeed. This highlights the difficulty in and importance of replicating scientific experiments.

## Design of new TMS structures

### Iteration 1

*Design*

To test the possibility of changing the D-loop of [BBa\_K4811002](http://parts.igem.org/Part%3ABBa_K4811002), we wanted to use an aptamer that was well characterised. The RNA-aptamer from Suess *et al.* (2004) is a well-characterised aptamer that binds to theophylline. To have the least conformational change in the TMS, we decided to incorporate the aptamer in the D-loop with the stem.

*Build & Test*

For the assembly, we ordered the whole construct from IDT as a gBlock and USER cloned it into the pUC19 backbone. After transforming the plasmids into *E. coli* BL21(DE3), we first tested whether the change in the TMS had altered the binding to RBS. Using varying concentrations of IPTG with a constant concentration of arabinose, we could see that the change in TMS did not alter the binding to the RBS. Furthermore, we then tested the system with varying concentrations of theophylline, and fluorescence decreased proportionally to its concentration. This however indicated that the TMS had a better binding to the RBS in the presence of theophylline.

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/theo2-tms.png)

Figure 6: OD normalized relative mCherry fluorescence, 561 nm excitation/617 nm emission, as a function of theophylline concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture with no IPTG or L-arabinose (uninduced).

*Learn*

These results were not as expected. We observed that increasing amount of ligand seemed to decrease fluorescence. This led us to consider if it was because of the insert design or the aptamers affinity to theophylline. To test one of the options, we decided to try a different insert.

### Iteration 2

*Design*

To try another insert, Suess *et al.* (2004) varied the length of the fragment surrounding the aptamer sequence. This made us question whether the stem of the D-loop was necessary. We designed a new TMS that did not include the stem to test this.

*Build & Test*

Without altering the conditions for the experiment, we executed it with the new TMS. The results also showed a decrease in fluorescence when we increased the concentration of theophylline.

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/theo1.png)

Figure 7: OD normalized relative mCherry fluorescence, 561 nm excitation/617 nm emission, as a function of theophylline concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture with no IPTG or L-arabinose (uninduced).

*Learn*

Comparing the result from the first iteration, the removal of the stem in the D-loop did not alter the tendency, where we saw an increased binding affinity to the RBS with higher concentration of theophylline. Yet, we did see that including the stem did improve the binding of the TMS to the RBS. Due to this unexpected result, we tried to use a completely different aptamer to test what the modification of the D-loop would cause.

### Iteration 3

*Design*

To learn more about how alterations in the TMS could influence the system, we wanted to try with a different aptamer. The aptamer for manganese from Dambach *et al.* (2015) has a similar size as the GFP aptamer, making it a good option to incorporate. It is also present in an existing biobrick, [BBa\_K902074](http://parts.igem.org/Part%3ABBa_K902074).

*Build & Test*

We ordered the TMS construct from IDT and USER cloned it into the pUC19 backbone. After transforming the two plasmids into BL21, we tested the system with varying concentrations of manganese. However, we saw high fluorescence when we used 0 µM of manganese.

As with theophylline, we created two different versions in the end: one with a longer stem region, TMS(Mn1), and one with only the manganese-binding part of the aptamer, TMS(Mn2).

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/mn1-tms.png)

Figure 8: OD normalized relative mCherry fluorescence, 561 nm excitation/617 nm emission, as a function of manganese concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture with no IPTG or L-arabinose(uninduced).

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/mn2-tms.png)

Figure 9: OD normalized relative mCherry fluorescence, 561 nm excitation/617 nm emission, as a function of manganese concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture with no IPTG or L-arabinose(uninduced).

*Learn*

The fluorescence from manganese could be from the manganese in the LB media. To change this we would have to have a different media, but as manganese is an important cofactor for enzymes, it is difficult to avoid in the experiment.

## Design of PFOA TMS

### Iteration 1

*Design*

The next step was to test aptamers for PFOA, designed by Park *et al.* (2022) using SELEX. They are not as well-characterized as the previous aptamers. The incorporation of the PFOA aptamer into the D-loop could create a TMS system sensitive for PFOA. The initial design would incorporate the whole PFOA aptamer with minimal changes, [BBa\_K4811024](http://parts.igem.org/Part%3ABBa_K4811024), only removing parts of the stem that seems to be less relevant for PFOA binding.

*Build & Test*

We procured the entire construct as a gBlock from IDT, and it was subsequently USER cloned into the pUC19 backbone. Following the transformation of these plasmids into *E. coli* BL21(DE3), our experiment aimed to determine whether the alteration in the TMS had any impact on its binding to the RBS. By employing varying concentrations of IPTG while keeping the arabinose concentration constant, we observed that the addition of the new D-loop to the TMS did not affect its binding to the RBS.

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/pfoa1-tms-induction.png)

Figure 10: mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of IPTG concentration. As control there is a culture with no IPTG or L-arabinose(uninduced)

Subsequently, we conducted experiments with varying PFOA concentration, where we observed the mCherry fluorescence decreased when the PFOA concentration increased.

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/pfoa1-tms.png)

Figure 11: mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of PFOA concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG (IPTG), one only induced by 0.1 % w/v L-arabinose (L-ara), and one with no inducers present (uninduced).

*Learn*

Once again, the observed effect was unexpected, with higher PFOA concentrations leading to lower fluorescence. This raised the question of whether the aptamer insert could be designed differently. Taking the same approach as with theophylline, we shortened the insert down to only contain a short stem and the aptamer.

### Iteration 2

*Design*

The new insert was designed to include the stem of the D-loop, [BBa\_K4811030](http://parts.igem.org/Part%3ABBa_K4811030). This new design removed the first 6 bp and the last 7 bp, reducing the insert from 45 bp to 32 bp.

*Build & Test*

The complete construct was ordered as a gBlock, which was USER cloned into pUC19 backbone. The experiment with varying PFOA concentration was performed. The same trend as in the previous iteration was observed in which high PFOA concentrations resulted in lower fluorescence levels.

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/pfoa2-tms.png)

Figure 12: mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of PFOA concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG (IPTG), one only induced by 0.1 % w/v L-arabinose (L-ara), and one with no inducers present (uninduced).

*Learn*

As with PFOA1, the trend showed lower mCherry fluorescence upon higher PFOA concentrations, but now the results were statistically significant. Seeing that restricting the aptamer to include less of the stem, we then went on to restrict the aptamer insert even more, only including the part of the aptamer that was predicted to change conformation upon binding to PFOA.

### Iteration 3

*Design*

The last design of the aptamer insert in [BBa\_K4811025](http://parts.igem.org/Part%3ABBa_K4811025) only includes 19 bp of the original aptamer, primarily the part predicted to be sensitive to PFOA.

*Build & Test*

The entire construct was acquired as a gBlock and subsequently USER cloned into the pUC19 backbone. After transformation, we used the same setup as for TMS(PFOA1) to confirm that the change did not influence the TMS function across IPTG concentrations, despite the aptamer insert being altered significantly.

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/pfoa3-tms-induction.png)

Figure 13: mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of IPTG concentration. As control there is a culture with no IPTG or L-arabinose(uninduced).

The obtained results confirmed this, with this mCherry fluorescence decreasing based on PFOA concentration. Therefore, the TMS system was tested using varying PFOA concentrations. This experiment showed the same trend of a decrease in fluorescence when PFOA concentration increased.

![](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/pfoa3-tms.png)

Figure 14: mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of PFOA concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG (IPTG), one only induced by 0.1 % w/v L-arabinose (L-ara), and one with no inducers present (uninduced).

*Learn*

After three iterations of the engineering cycle, we have learned that the change in the size of the aptamer does not have an effect on the way PFOA interacts with our TMS. The observed trend remains similar throughout all three aptamer iterations. However, the strength of the interaction does vary. As the D-loop decreases in size, it is harder to destabilize the interaction between the TMS and our reporter gene. Thus, the smallest design is the one that reduces the amount of fluorescence to the smallest degree.

The largest fold change in mCherry fluorescence upon addition of increasing PFOA concentrations was seen in TMS(PFOA2). Though this is not the result we expected, it is still very useful and does show mCherry fluorescence dependent on the concentration of PFOA.

## Design of a cell-free system

### Iteration 1

*Design*

Cell-free systems (CFS) are a solution that mimic cell behavior. In particular, transcription and translation CFS (TXTL-CFS) are able to drive the protein synthesis, both of linear and circular DNA. The furthest extent of our project, a technology based on a TMS system that is able to reliably and reproducibly detect PFOA in water, would ideally work outside of the cellular environment to overcome some of the main limitations common to all synthetic biology applications.

The use of a TXTL-CFS allows the production of a PFOA detection device that can be produced on a large scale without the need for rare compounds or electronic components. It can be stored dry up to months and is activated upon rehydration. In our concept, rehydration with field-collected water samples would activate the mRNA transcription and translation of the reporter protein, giving a detectable signal after a few hours of room temperature incubation.

DTU Bioengineering has a biophysics group that works with cell-free systems. We had a meeting with Rasmus Krogh Norrild from the group to discuss the best way to design the system.

*Build & Test*

We unfortunately did not get to test our design or further develop it.

## References

* Dambach, M., Sandoval, M., Updegrove, T. B., Anantharaman, V., Aravind, L., Waters, L. S., & Storz, G. (2015). The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element. Molecular cell, 57(6), 1099–1109. <https://doi.org/10.1016/j.molcel.2015.01.035>
* Park, J., Yang, K. A., Choi, Y., & Choe, J. K. (2022). Novel ssDNA aptamer-based fluorescence sensor for perfluorooctanoic acid detection in water. Environment international, 158, 107000. <https://doi.org/10.1016/j.envint.2021.107000>
* Suess, B., Fink, B., Berens, C., Stentz, R., & Hillen, W. (2004). A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic acids research, 32(4), 1610–1614. <https://doi.org/10.1093/nar/gkh321>

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

# Experiments

Photo: DTU

##### Menu

* [> Weekly Laboratory Notebook](#weekly_lab)
* [> Protocols](#protocols)

## Weekly Laboratory Notebook

This page contains the laboratory work conducted by DTU Biobuilders in the period June 19th, 2023, to October 8th, 2023. Weekly summaries can be found in the **Weekly Laboratory Notebook**, and the full Benchling can be found [here](https://static.igem.wiki/teams/4811/wiki/data/general-eln-benchling-compressed.pdf). All work was done according to the protocols described on this page. Please click on the week you would like to check in the table below:

|  |  |  |  |
| --- | --- | --- | --- |
| Week1(19/06/23-25/06/23) | Week5(17/07/23-23/07/23) | Week9(14/08/23-20/08/23) | Week13(11/09/23-17/09/23) |
| Week2(26/06/23-02/07/23) | Week6(24/07/23-30/07/23) | Week10(21/08/23-27/08/23) | Week14(18/09/23-24/09/23) |
| Week3(03/07/23-09/07/23) | Week7(31/07/23-06/08/23) | Week11(28/08/23-03/09/23) | Week15(25/09/23-01/10/23) |
| Week4(10/07/23-16/07/23) | Week8(07/08/23-13/08/23) | Week12(04/09/2023-10/09/2023) | Week16(02/10/2023-08/10/2023) |

### Week 1

We prepared competent DH5α cells according to the protocol **Chemical competent cells**. These were to be used in upcoming experiments.

We also received primers ordered and prepared these. gBlocks were ordered from IDT, and these were PCR amplified using Taq polymerase following the **protocol from Ampliqon**. Only weak bands were seen on the gel, so it was decided to do the experiment again.

### Week 2

gBlocks were successfully PCR amplified using Taq polymerase following the **protocol from Ampliqon**. Products were stored in the freezer.

We prepared two of our backbones by linearizing pUC19. This was done following the **protocol for X7 PCR** using Phusion buffer instead of CxL. Fragments were checked on a gel and purified from overnight cultures the following day. The plasmid purification was conducted following the **protocol from Invitrogen**.

To obtain the second plasmid we needed for our experiments, we transformed pACYC184 into DH5α. Growth was obtained in LB supplemented with chloramphenicol. Concentrations of pACYC184 were too low to be visualized on a gel with EtBr. It was concluded that a PCR had to be done. After 2 days of growth, we were able to purify the plasmids using the **Monarch® Plasmid Miniprep Kit from NEB**. The obtained concentrations were rather low, so PCR amplification was conducted.

DH5α was transformed with GFP tet-on and mCherry reporter. Transformants were plated on LB supplemented with kanamycin. Controls were made in duplicates as follows: DH5α plated directly on LB with kanamycin, and DH5α plated directly on LB. Growth occurred for DH5α on LB and transformed DH5α on LB with kanamycin. The transformation was considered successful.

We also wanted to test the GFP tet-on system in DH5α. We inoculated 1 colony in 1 mL LB supplemented with tetracycline. It was left to incubate overnight at 37°C.

PCR was conducted on our 6 TMS’s (Theo1, Theo2, Kan, Neo, GFP, Mn) following the **protocol for X7 PCR**.

### Week 3

Troubleshooting PCRs by testing three different X7 polymerases on the TMS aptamer with GFP using appropriate primers. The **protocol for X7 PCR** was followed. Buffer used was NEB 2x mastermix. Only faint bands were obtained.

First attempt to make competent BL21(DE3) cells was conducted following **Chemical competent cells**. Due to low growth, this was postponed to next week.

Initial fluorescence measurement of ordered plasmids containing mCherry and GFP-teton. Inducers for the two plasmids were arabinose and tetracycline, respectfully. Different concentrations of the inducers were added:

|  |  |  |  |
| --- | --- | --- | --- |
| Arabinose (15 g/L) |  | Tetracycline (10 mg/mL) |  |
| Concentration (g/100 mL) | μL added | Concentration (μg/mL) | μL added |
| 0 | 0 | 0 | 0 |
| 0.05 | 16.7 | 0.5 | 0.25 |
| 0.1 | 33 | 1 | 0.5 |
| 0.3 | 100 | 5 | 2.5 |

The strains harboring the plasmids were tested on Fluorescence Spectrometer JASCO model number FP-8500 with GFP emission measured by excitation at 395 nm, mCherry emission measured by excitation at 561 nm. The presence of GFP was shown, but the presence of mCherry could not be seen. This, we found out was because we were using D-arabinose, which is unable to induce the araBAD promoter.

First successful USER cloning of the TMS-GFP into pUC19 (pU07) following the protocol **USER cloning**. These were tested the following week.

### Week 4

This week we prioritized: Troubleshooting PCRs, USER cloning of our plasmid, and looking into further fluorescence measurements.

New competent BL21(DE3) were made and their competency checked using pUC19 control from NEB. Growth of transformed strains was not obtained on LB supplemented with ampicillin plates, but this was also not achieved for our competent DH5α. Growth on LB plates was obtained.

We continued our experiments for mCherry induction by arabinose. Here, we came to understand that our araBAD promoter is only induced by L-arabinose, and we had been using D-arabinose. We fixed the problem and obtained some initial results:

| Arabinose induction (g/L) | Fluorescence | OD600 |
| --- | --- | --- |
| 0 | 168 | 0.197 |
| 0.3 | 449 | 0.512 |
| 0.7 | 638 | 0.553 |
| 1 | 627 | 0.507 |

For troubleshooting PCRs, we tried to add DMSO, reduce the primer concentration, and change the annealing temperature. No additional knowledge was achieved from this.

We successfully USER cloned pU08-10, but not pU02. These were checked with **colony PCR** in the following week.

### Week 5

BL21(DE3) competency test by transforming the competent cells with pACYC184, pUC19 (kindly provided by Mogens Kilstrup), and pUC19 (NEB), respectively. A control with MQ was also made and plated onto LB. Transformations were done according to **Transformation of *E. coli*** (normal heating times used, and for pACYC184, cells were incubated in 475 μL LB at 37°C for 1 hour). Growth was obtained on all plates, thus, cells were concluded to be competent.

New batch of competent DH5α was made by following **Chemical competent cells**. Cells were stored in the -80°C freezer.

Our USER constructs, pU08-10, were checked with **colony PCR** (elongation time of 2.5 min). Overnight cultures were made of the validated strains, and the plasmids were purified using **Monarch® Plasmid Miniprep Kit from NEB**, and cryostocks were made.

We continued with our PCRs to amplify the following fragments: B01, B03, B07, B11, B12, B13. We used the PCR program with annealing temperature of 57°C and elongation time of 30 s for B01 and B03, and annealing temperature of 56°C and elongation time of 2.5 min for B07, B12, and B13.

PCR troubleshooting: we figured that we had been using an unfit buffer (10x buffer), we then decided to use rCutSmart moving on. Also concluded that 2X Phusion U Hot Start Master Mix from Thermo Fisher could be used.

USER clonings of pU01, pU02, and pU03. The USER cloning was conducted according to **USER cloning**, and all were grown in LB for 1 hour before plated onto LB supplemented with chloramphenicol and ampicillin. The positive control was pACYC184, and the negative control was the backbone (G03) and MQ. Nice positive and negative controls were obtained, but no colonies on the cloned plates.

### Week 6

PCRs of B10, B11, B12, and G03 were redone using rCutSmart as buffer. Also, B03, G05, G06, G08, B07, and B09. Gel extraction was done on B07, G05, G06, G08, and PCR cleanup on B03 and B09. Concentrations and purities were the best for B03 and B09, and moving on we used **PCR cleanup** instead of gel extraction.

USER clonings of pU01, pU02, and pU03 were redone. We had three versions of the backbone (G03), and **USER cloning** was conducted with one of each doing equimolar amounts of backbone and fragment. As a positive control pACYC184 was used, and cloning was done with 1 min of heat shock, and incubation in 450 μL LB media for 1 hour before plating onto LB plates supplemented with chloramphenicol.

The cloning did not succeed. We considered if this was due to the plasmid backbone not being opened correctly. A possible reason for this could be plasmid supercoiling for which reason linearization using restriction enzymes would be the best option. Another cause could be the annotated plasmid pACYC184 is not correct.

An experiment was set up to assess the linearized backbone issue. We wanted to check if the previously excised band was just a supercoiled topoisomer. A gel was run with two different PCR mixes for p03 reaction against the non-linearized plasmid. PCRs were done with Phusion MM2X HotStart and X7-CutSmart. Unfortunately, the extension time was not enough for the X7 PCR (2.5min, should have been 3.5min), and on the gel the bands looked more lightweight than they should; nonetheless it was visible that the amplified band ripple, as if there were more PCR products than just the expected one, and the rippling pattern did not correspond to the pattern of the nonlinearized backbones, which is likely only due to just supercoiling. The Phusion reaction did show a band of the right size, which was surrounded up and down by thinner bands corresponding to the nonlinearized backbone supercoiled ripples.

Another Phusion was done, which gave a nice, clear band. This was cut out from the gel and extracted. Due to leftover salts a cleanup was attempted following the second half of a plasmid prep protocol (invitrogen).

We proceeded with **USER cloning** and did pU01. Colonies were obtained, and transformants were validated using **colony PCR**. Overnight cultures were prepared for next week.

### Week 7

Plasmid pU01, which was made and validated the week before, was purified and a glycerol stock was made. The purified plasmid was additionally checked in a restriction enzyme assay using the restriction enzymes SspI and AdhI. AdhI should generate two fragments of 1054 bp and 4212 bp, and SspI three fragments of 873 bp, 1728 bp, and 2665 bp.

pU01 was transformed into BL21(DE3) to generate a reporter strain, which we used for assessing mCherry production upon arabinose induction. Thus, the strain was induced with varying concentrations of arabinose. The tested concentrations were in g/L: 0, 0.1, 0.2, 0.3, 0.5, 0.7, and 1. Fluorescence increased with increased arabinose concentrations, from 112 to 383, and a fluorescence of 92 for the negative control.

The plasmid backbone pUC19 with kanamycin resistance was constructed with USER cloning using an amplified kanamycin resistance cassette and linearized pUC19 without the ampicillin resistance. The cloned strains were validated with **colony PCR** and restriction enzyme assay. The restriction enzyme used was SspI, since this generates two distinct fragments (size 374 bp and 2276 bp) and also cuts within the kanamycin resistance cassette. Correct plasmids were purified and glycerol stocks were made.

We attempted double transformations with the USER cloned plasmids pU07-pU10 into BL21(DE3). Due to insufficient transformations a new protocol from [Eindhoven iGEM 2014](https://static.igem.org/mediawiki/2014/9/94/TU_Eindhoven_Protocol_Double_Transformation.pdf). No successful double transformations were achieved this week.

MIC test of BL21(DE3) with tetracycline. From the results, it seemed cells started to grow at a concentration of 0.2 μg/mL and can survive at concentrations below that.

### Week 8

Transformation of USER-cloned plasmids into BL21(DE3). We had trouble getting two plasmids into one strain.

* Unsuccessful transformations tried: BL21(DE3) + pU07 + pU01, BL21(DE3) + pU07 + 2xpU01, BL21(DE3).pU01 + pU07.
* Successful transformations: BL21(DE3).pU01, BL21(DE3), BL21(DE3) + pU07.

To test why the double transformations were unsuccessful, different combinations of transformations were done following the protocol from [Eindhoven](https://static.igem.org/mediawiki/2014/9/94/TU_Eindhoven_Protocol_Double_Transformation.pdf). Additionally, transformations with a 1 hour induction and a 2 hour induction were conducted. The following transformation combinations were tried: BL21(DE3), BL21(DE3) + pU01 + pU07, BL21(DE3).pU01, BL21(DE3).pU01 + pU07, BL21(DE3).pU07, BL21(DE3).pU07 + pU01. Combinations with “+” are transformed at the same time, and transformations with “.” are done as two rounds of transformation. Transformants were plated on four different plates: LB, LB supplemented with ampicillin, LB supplemented with chloramphenicol, and LB supplemented with ampicillin and chloramphenicol. All transformants were plated on all the different plates, except BL21(DE3) containing both plasmids was not plated onto LB only plates.

One successful transformation of BL21(DE3).pU01.pU07 was obtained. Some varying results were obtained from the 2 hour induction transformants, and these transformations were therefore redone. Alongside, transformations with pU08, pU09, and pU10 were also done.

PCR of B01 and B03. The PCR was done according to **X7 PCR** with an elongation time of 12 sec, since the two fragments have the size of 175 bp and 173 bp, respectively. The PCR products were cleaned up using **Ampliqon PureIT ExoZAP PCR CleanUp** and following the instructions given by the manufacturer. The products were verified on a gel and concentrations and purities were evaluated on a Nanodrop.

USER cloning of pU05 and pU06 using G02 as the vector backbone and B01 and B03 in each construct, respectively. The protocol followed was **USER cloning**. As a negative control MQ was used, and pUC19 (KanR) was used as a positive control. Before plating was done on LB supplemented with kanamycin, the transformants were incubated in LB for 2 hours at 37°C. The following day, all plates contained transformants, and the cloning was therefore attempted again to see if this was just a mistake or something was off with the backbone. The repetition gave the same results, so it was decided to do **colony PCR** on both rounds of transformants. The cPCR was done using Taq polymerase. These results were not convincing.

We tested tetracycline and arabinose induction of the TMS + mCherry system, the one in BL21(DE3).pU01. Cells were grown until they reached an OD of 0.3, after which they were divided into Eppendorfs and 0.5 g/L arabinose and varying concentrations of tetracycline (listed in table) was added. Cells were incubated at 37°C for 5 hours, and fluorescence was measured on the plate reader BioTek Cytation 5.

| Tetracycline (μg/mL) | Tetracycline concentration (μg/mL) | Tetracycline (μL) | Arabinose (g/L) | Arabinose (mL) | LB (mL) | Final volume (mL) | Arabinose concentration (g/L) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0.75 | 10 | 37.5 | 0.5 | 5 | 457.5 | 0.5 | 50 |
| 0.375 | 10 | 18.75 | 0.5 | 5 | 476.25 | 0.5 | 50 |
| 0.1875 | 10 | 9.375 | 0.5 | 5 | 485.625 | 0.5 | 50 |
| 0.09375 | 10 | 4.6875 | 0.5 | 5 | 490.3125 | 0.5 | 50 |
| 0.046875 | 10 | 2.34375 | 0.5 | 5 | 492.65625 | 0.5 | 50 |
| 0.0234375 | 10 | 1.171875 | 0.5 | 5 | 493.828125 | 0.5 | 50 |
| 0 | 0 | 0 | 0.5 | 5 | 495 | 0.5 | 50 |

After 5 hours, the following results were obtained:

| Tetracycline (μg/mL) | Fluorescence |
| --- | --- |
| 0.75 | 67 |
| 0.375 | 201 |
| 0.1875 | 170 |
| 0.09375 | 230 |
| 0.046875 | 187 |
| 0.0234375 | 191 |
| 0 | 174 |

### Week 9

Another cPCR of pU05 and pU06 transformants. This time following

X7 PCR

. PCR products were run on a gel, but no bands were visible for pU05, some vague bands around the right size were visible for pU06.

We sent our constructed plasmids to sequencing by Eurofins. The following was sent with their respective primers: pU01, pU07, pU08, pU09, pU10, and pUC19 (kanR). For some of the plasmids, more than one copy was sent.

BL21(DE3).pU01 was made competent, as we considered this a way to increase the chances of successful double transformations. We followed the protocol **Chemical competent cells**.
More competent DH5α was also made following the same protocol.

Another attempt of USER cloning of pU05 and pU06 was done alongside USER clonings of pU02, pU03, and pU04. Cloning was done following **USER cloning protocol**. Colony PCR was done the following week.

### Week 10

Received sequencing data from Eurofins. pU07, pU09 seemed correct.

We did some new **colony PCR** using Taq polymerase on pU03, pU04, pU05, and pU06. Some of the pU03 had the correct band size, whereas all pU04 had one extra band. It was also concluded that one of the pU06 could be correct, whereas no bands were seen for pU05. Overnight cultures were made of correct plasmids, and these were then purified the following day using the **Monarch® Plasmid Miniprep Kit from NEB** and following the instructions given by the manufacturer.

More plasmids were sent for sequencing with appropriate primers. These were: pU01, pU03, pU08, and pU10.

This week we also received competent BL21(DE3) (C2527H) from NEB, and with these we initially tried another double transformation. We transformed pU01 and pU08 into the competent cells following the **protocol plasmid transformation in *E. coli***, using 2 μL of each plasmid with a concentration of 2 ng/μL. The cells were heat shocked for 50 s and plated onto LB supplemented with chloramphenicol and ampicillin. The following day plenty of colonies were obtained and these were checked with **colony PCR**. All checked colonies showed the right band length. Two glycerol stocks were made.

We then proceeded with the other double transformations and did BL21(DE3).pU01.pU09, BL21(DE3).pU01.pU10, and BL21(DE3).pU03.pU07. BL21(DE3).pU03.pU07 was not plated since a sudden lack of plates occurred. The others were plated, and transformants were checked the following week.

### Week 11

BL21(DE3).pU01.pU09 and BL21(DE3).pU01.pU10 from the week before were checked with **colony PCR** using Taq polymerase. Overnight cultures for cryostocks were made.

Sequencing data was received. Everything, but pU06, looked as expected.

More double transformations were done using the competent BL21(DE3) from NEB. We proceeded with BL21(DE3).pU03.pU07 and BL21(DE3).pU01.pU06. Since we figured out pU06 was wrong, the latter transformant was discarded. Transformation was done following the **protocol plasmid transformation in *E. coli***, using 2 μL of each plasmid with a concentration of 2 ng/μL, and a heat shock of 50 s. Here, no colonies were obtained.

We began fluorescent measurement on the strains we had constructed this far: BL21(DE3).pU01, BL21(DE3).pU01.pU08, BL21(DE3).pU01.pU09, and BL21(DE3).pU01.pU10. OD from overnight cultures was low, so instead of making a new inoculation in the morning, we induced the overnight cultures directly according to the schemes below.

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| BL21(DE3).U01.U07 + arabinose (0.1% (w/v)), varying IPTG (mM) | 0 | 0.1 | 0.5 | 1 | 2 | 5 | Blank |
| BL21(DE3).U01.U07 + 1 mM IPTG, varying arabinose (% (w/v)) | 0 | 0.01 | 0.05 | 0.1 | 0.2 |  | Blank |
| BL21(DE3).U01.U08 + Mn (μM) | 0 | 0.1 | 1 | 10 | 100 | 1000 |  |
| BL21(DE3).U01.U09 + Theophylline (mM) | 0 | 0.1 | 1 | 2 | 5 | 10 |  |
| BL21(DE3).U01.U10 + Theophylline (mM) | 0 | 0.1 | 1 | 2 | 5 | 10 |  |

BL21(DE3).pU01.pU07 + arabinose (0.1 % (w/v)), varying IPTG (mM)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| [IPTG] (mM) | E. coli (μL) | v(IPTG) (μL) | v(MQ) (μL) | Arabinose (μL) |
| Blank (0) | 250 | 0 | 250 | 0 |
| 0 | 250 | 0 | 240 | 10 |
| 0.1 | 250 | 0.5 | 239.5 | 10 |
| 0.5 | 250 | 2.5 | 237.5 | 10 |
| 1 | 250 | 5 | 235 | 10 |
| 2 | 250 | 10 | 230 | 10 |

BL21(DE3).pU01.pU07 + 1 mM IPTG (mM), varying L-arabinose (% (w/v))

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| [Arabinose] (% w/v) | [Arabinose] (g/L) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) |
| Blank (0) | 0 | 250 | 0 | 250 | 0 |
| 0 | 0 | 250 | 0 | 245 | 5 |
| 0.1 | 1 | 250 | 10 | 235 | 5 |
| 0.5 | 5 | 250 | 50 | 195 | 5 |
| 1 | 10 | 250 | 100 | 145 | 5 |
| 2 | 20 | 250 | 200 | 45 | 5 |

BL21(DE3).pU01.pU08 + manganese (μM)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| [MnCl2] (μM) | E. coli (μL) | v(MnCl2) (μL) | v(MQ) (μL) | Arabinose (μL) | IPTG (μL) |
| 0 | 250 | 0 | 235 | 10 | 5 |
| 0.1 | 250 | 25 | 210 | 10 | 5 |
| 1 | 250 | 2.5 | 232.5 | 10 | 5 |
| 10 | 250 | 25 | 210 | 10 | 5 |
| 100 | 250 | 2.5 | 232.5 | 10 | 5 |
| 1000 | 250 | 25 | 210 | 10 | 5 |

BL21(DE3).U01.U09 + theophylline (mM)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| [Theophylline] (mM) | E. coli (μL) | v(theophylline) (μL) | v(MQ) (μL) | Arabinose (μL) | IPTG (μL) |
| 0 | 250 | 0 | 235 | 10 | 5 |
| 0.1 | 250 | 2 | 235 | 10 | 5 |
| 1 | 250 | 20 | 215 | 10 | 5 |
| 2 | 250 | 40 | 195 | 10 | 5 |
| 5 | 250 | 100 | 135 | 10 | 5 |
| 10 | 250 | 200 | 35 | 10 | 5 |

BL21(DE3).U01.U10 + theophylline (mM)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| [Theophylline] (mM) | E. coli (μL) | v(theophylline) (μL) | v(MQ) (μL) | Arabinose (μL) | IPTG (μL) |
| 0 | 250 | 0 | 235 | 10 | 5 |
| 0.1 | 250 | 2 | 235 | 10 | 5 |
| 1 | 250 | 20 | 215 | 10 | 5 |
| 2 | 250 | 40 | 195 | 10 | 5 |
| 5 | 250 | 100 | 135 | 10 | 5 |
| 10 | 250 | 200 | 35 | 10 | 5 |

Measurements after 5 hours were done on a plate reader BioTek Cytation 5.

### Week 12

**Colony PCR** of BL21(DE3).pU03.pU07 was conducted. Correctly looking plasmids were **purified** and their concentration and purity were checked on a Nanodrop. Additionally, cryostocks were made.

We prepared overnight cultures of BL21(DE3).pU03.pU07 to be used the following week in a fluorescent measurement.

### Week 13

Fluorescent measurements were done on BL21(DE3).pU03.pU07. We did the same measurement twice this week.

An overnight culture was inoculated to an OD of 0.05 in LB supplemented with ampicillin and chloramphenicol. After 2 hours an OD around 0.3-0.6 was obtained. Induction was done according to the scheme below using the stock solutions 50 g/L L-arabinose and 100 mM IPTG, the second time a stock of 25 g/L L-arabinose was used for the varying arabinose measurements.

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0 | 0.5 | 1 | 2 | 4 | 6 | 8 | 10 | Blank |
| 0 | 0.5 | 1 | 2 | 4 | 6 | 8 | 10 | Blank |
| 0 | 0.5 | 1 | 2 | 4 | 6 | 8 | 10 | Blank |
| 0 | 0.5 | 1 | 2 | 4 | 6 | 8 | 10 | Blank |
| 0 | 0.005 | 0.01 | 0.03 | 0.05 | 0.1 | 0.25 | 0.5 | Blank |
| 0 | 0.005 | 0.01 | 0.03 | 0.05 | 0.1 | 0.25 | 0.5 | Blank |
| 0 | 0.005 | 0.01 | 0.03 | 0.05 | 0.1 | 0.25 | 0.5 | Blank |
| 0 | 0.005 | 0.01 | 0.03 | 0.05 | 0.1 | 0.25 | 0.5 | Blank |

| [IPTG] (mM) | E. coli (μL) | v(IPTG) (μL) | v(MQ) (μL) | Arabinose (μL) |
| --- | --- | --- | --- | --- |
| 0 | 100 | 0 | 98 | 2 |
| 0.5 | 100 | 1 | 97 | 2 |
| 1 | 100 | 2 | 96 | 2 |
| 2 | 100 | 4 | 94 | 2 |
| 4 | 100 | 8 | 90 | 2 |
| 6 | 100 | 12 | 86 | 2 |
| 8 | 100 | 16 | 82 | 2 |
| 10 | 100 | 20 | 78 | 2 |
| Blank | 100 | 0 | 100 | 0 |

| [Arabinose] (% w/v) | [Arabinose] (g/L) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 0 | 100 | 0 | 100 | 0 |
| 0.005 | 0.05 | 100 | 0.4 | 99.6 | 0 |
| 0.01 | 0.1 | 100 | 0.8 | 99.2 | 0 |
| 0.03 | 0.3 | 100 | 2.4 | 97.6 | 0 |
| 0.05 | 0.5 | 100 | 4 | 96 | 0 |
| 0.1 | 1 | 100 | 8 | 92 | 0 |
| 0.25 | 2.5 | 100 | 20 | 80 | 0 |
| 0.5 | 5 | 100 | 40 | 60 | 0 |
| Blank | 0 | 100 | 0 | 100 | 0 |

Measurements after 5 hours were done on the plate reader BioTek Cytation 5.

### Week 14

USER cloned pU11-14 following **USER cloning protocol** using 5 μL BioBrick DNA and 1 μL USER-ready vector. Cloned mixture was left at 30°C for 20 min, 20°C for 20 min, and 12°C for 10 min. They were then transformed into competent DH5α cells according to the **Transformation in *E. coli*** protocol, and plated onto LB supplemented with ampicillin.

The transformants were genotyped with **colony PCR** using appropriate primers and following **X7 PCR**. Cells were diluted in 20 μL MQ, and incubated at 80°C for 5 min. The elongation time was set to 30 s, since the expected band length was 300-400 bp. The PCR products were checked on a gel, and all colonies seemed correct. Overnight cultures of two of each plasmid were made using LB supplemented with ampicillin. The plasmids were purified. Concentrations and qualities were checked on a Nanodrop.

We did another fluorescence test with BL21(DE3).pU01.pU08, BL21(DE3).pU01.pU09, BL21(DE3).pU01.pU10. The same setup as previously was applied. From overnight cultures, the cells were inoculated to OD 0.1, and left to grow for 2 hours in LB supplemented with ampicillin and chloramphenicol. Stock solutions used were 100 mM IPTG, 50 g/L L-arabinose, and 25 g/L L-arabinose.

Using a 100 mM IPTG stock and 50 g/L L-arabinose.

| [IPTG] (mM) | E. coli (μL) | v(IPTG) (μL) | v(MQ) (μL) | Arabinose (μL) |
| --- | --- | --- | --- | --- |
| 0 | 100 | 0 | 96 | 4 |
| 0.5 | 100 | 1 | 95 | 4 |
| 1 | 100 | 2 | 94 | 4 |
| 2 | 100 | 4 | 92 | 4 |
| 4 | 100 | 8 | 88 | 4 |
| 6 | 100 | 12 | 84 | 4 |
| 8 | 100 | 16 | 80 | 4 |
| 10 | 100 | 20 | 76 | 4 |
| Blank | 100 | 0 | 100 | 0 |

Using a 25 g/L L-arabinose stock.

| [Arabinose] (% w/v) | [Arabinose] (g/L) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 0 | 100 | 0 | 100 | 0 |
| 0.005 | 0.05 | 100 | 0.4 | 99.6 | 0 |
| 0.01 | 0.1 | 100 | 0.8 | 99.2 | 0 |
| 0.03 | 0.3 | 100 | 2.4 | 97.6 | 0 |
| 0.05 | 0.5 | 100 | 4 | 96 | 0 |
| 0.1 | 1 | 100 | 8 | 92 | 0 |
| 0.25 | 2.5 | 100 | 20 | 80 | 0 |
| 0.5 | 5 | 100 | 40 | 60 | 0 |
| Blank | 0 | 100 | 0 | 100 | 0 |

### Week 15

Double transformations pU11-14 into competent BL21(DE3) from NEB with pU01. Following the **protocol Transformation in *E. coli***. Transformants were plated onto LB supplemented with ampicillin and chloramphenicol. They were left to grow overnight at 37°C. First round of transformations gave no colonies, so a new double transformation was conducted. Using 2 μL of 2 ng/μL plasmids, and following the protocol **Plasmid transformation of *E. coli***, but with 50 s heat shock. Plating 200 μL of the transformants onto relevant media, and left overnight at 37°C.

Colonies were obtained for BL21(DE3).pU01.pU11 and BL21(DE3).pU01.pU13, so these were checked with **colony PCR** using Taq polymerase. All looked good when run on a gel. Overnight cultures of these were prepared.

The double transformations were done again, since not all gave colonies. They were plated onto LB supplemented with ampicillin and chloramphenicol, and left overnight at 37°C.

pU11-14 was sent to sequencing at Eurofins following the instructions given by Eurofins and with appropriate primers. Results showed that the plasmids were as expected, except for an uninspected insert in the ori, but this could be due to our plasmid map not corresponding perfectly to our plasmid.

Fluorescence test of BL21(DE3).pU03.pU07 was attempted again. An overnight culture was inoculated to an OD 0.04, and left to grow for 2 hours. For the induction following stock solutions were used: 50 g/L L-arabinose stock, 100 mM IPTG stock, and 5 μM anhydrotetracycline (aTc).

The induction plate looked as follows:

**[columns 1-3]**: BL21(DE3).pU03.pU07 + L-arabinose (0.1 % (w/v)), varying IPTG (mM)

**[columns 4-6]**: BL21(DE3).pU03.pU07 + 0 mM IPTG, varying L-arabinose (% (w/v))

**[columns 7-9]**: BL21(DE3).pU03.pU07 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying aTc (uM)

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 0.5 | 0.5 | 0.5 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
| 1 | 1 | 1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| 4 | 4 | 4 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| 8 | 8 | 8 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank | Blank |
| LB | MQ | BL21(DE3) |  |  |  |  |  |  |  |  |  |

| [IPTG] (mM) | E. coli (μL) | v(IPTG) (μL) | v(MQ) (μL) | Arabinose (μL) |
| --- | --- | --- | --- | --- |
| 0 | 100 | 0 | 96 | 4 |
| 0.5 | 100 | 1 | 95 | 4 |
| 1 | 100 | 2 | 94 | 4 |
| 4 | 100 | 8 | 88 | 4 |
| 8 | 100 | 16 | 80 | 4 |
| Blank | 100 | 0 | 100 | 0 |

| [Arabinose] (% w/v) | [Arabinose] (g/L) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 0 | 100 | 0 | 100 | 0 |
| 0.01 | 0.1 | 100 | 0.4 | 99.6 | 0 |
| 0.1 | 1 | 100 | 4 | 96 | 0 |
| 0.5 | 5 | 100 | 20 | 80 | 0 |
| 1 | 10 | 100 | 40 | 60 | 0 |
| Blank | 0 | 100 | 0 | 100 | 0 |

| [aTc] (μM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | aTc (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.01 | 100 | 4 | 93.6 | 2 | 0.4 |
| 0.1 | 100 | 4 | 90 | 2 | 4 |
| 0.5 | 100 | 4 | 74 | 2 | 20 |
| 1 | 100 | 4 | 54 | 2 | 40 |
| Blank | 100 | 0 | 100 | 0 | 0 |

| [aTc] (μM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | aTc (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 10 | 70 | 20 | 0 |
| 0.01 | 100 | 10 | 69.6 | 20 | 0.4 |
| 0.1 | 100 | 10 | 66 | 20 | 4 |
| 0.5 | 100 | 10 | 50 | 20 | 20 |
| 1 | 100 | 10 | 30 | 20 | 40 |
| Blank | 100 | 0 | 100 | 0 | 0 |

After induction, cells were left to grow for 5 hours. After 5 hours OD and fluorescence was measured on the plate reader SpectraMax i3. Growth was insufficient, so the cells were left to grow further overnight at 37°C.

Another fluorescence test was done on the remaining TMS systems. That being, BL21(DE3).pU01.pU08, BL21(DE3).pU01.pU09, BL21(DE3).pU01.pU10, BL21(DE3).pU01.pU11, and BL21(DE3).pU01.pU13.

As controls, the following was used:

* BL21(DE3): background absorption/fluorescence of the cells.
* pU01: mCherry fluorescence in the low copy number plasmid (must be induced with L-arabinose).
* pIDT02: mCherry fluorescence in a high copy number plasmid (must be induced with L-arabinose).

We used the following stock solutions: 20 mM, 2 μM and 200 μM MnCl2 stock (tetrahydrate - 197.91 g/mol), 25 mM theophylline, 50 g/L L-arabinose, and 100 mM IPTG.

The induction plate looked as follows:

**[columns 1-3]**: BL21(DE3).pU01.pU08 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying Mn (uM)

**[columns 4-6]**: BL21(DE3).pU01.pU09 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying theophylline (mM)

**[columns 7-9]**: BL21(DE3).pU01.pU10 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying theophylline (mM)

**[columns 10-12]**: BL21(DE3).pU01.pU11 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying PFOA (mM)

**[row G-H]**: BL21(DE3).pU01.pU13 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying PFOA (mM)

Controls are (shown in blue):

LB: 200 μL LB

MQ: 200 μL MQ

BL21(DE3): 100 μL MQ + 100 μL BL21(DE3)

pIDT2 cryo: 96 μL MQ + 100 μL pIDT2 from cryostock (showed growth) + 4 μL L-arabinose (0.1% w/v)

pIDT2: 96 μL MQ + 100 μL pIDT2 from (showed no growth) + 4 μL L-arabinose (0.1% w/v)

pU01: 96 μL MQ + 100 μL pU01 (showed growth) + 4 μL L-arabinose (0.1% w/v)

BL21(DE3).pU01.pU08 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying manganese (uM).

| [Mn] (μM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | Mn (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.1 | 100 | 4 | 84 | 2 | 10 |
| 1 | 100 | 4 | 93 | 2 | 1 |
| 10 | 100 | 4 | 84 | 2 | 10 |
| 100 | 100 | 4 | 93 | 2 | 1 |
| Blank | 100 | 0 | 100 | 0 | 0 |
| Mn concentrations (μM) | 2 | 200 | 20000 |  |  |
| [IPTG] (mM) | 1 | [arabinose] (%w/v) | 0.1 |  |  |

BL21(DE3).pU01.pU09 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying theophylline (mM).

| [Theophylline] (mM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | Theophylline (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.1 | 100 | 4 | 93.2 | 2 | 0.8 |
| 1 | 100 | 4 | 86 | 2 | 8 |
| 4 | 100 | 4 | 62 | 2 | 32 |
| 10 | 100 | 4 | 14 | 2 | 80 |
| Blank | 100 | 0 | 100 | 0 | 0 |
| Theo stock concentration (mM) | 25 |  |  |  | 0 |
| [IPTG] (mM) | 1 | [arabinose] (%w/v) | 0.1 |  |  |

BL21(DE3).pU01.pU10 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying theophylline (mM).

| [Theophylline] (mM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | Theophylline (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.1 | 100 | 4 | 93.2 | 2 | 0.8 |
| 1 | 100 | 4 | 86 | 2 | 8 |
| 4 | 100 | 4 | 62 | 2 | 32 |
| 10 | 100 | 4 | 14 | 2 | 80 |
| Blank | 100 | 0 | 100 | 0 | 0 |
| Theo stock concentration (mM) | 25 |  |  |  | 0 |
| [IPTG] (mM) | 1 | [arabinose] (%w/v) | 0.1 |  |  |

BL21(DE3).pU01.pU13 + L-arabinose (0.1 % (w/v)), varying IPTG (mM).

| [IPTG] (mM) | E. coli (μL) | v(IPTG) (μL) | v(MQ) (μL) | Arabinose (μL) |
| --- | --- | --- | --- | --- |
| 0 | 100 | 0 | 96 | 4 |
| 0.5 | 100 | 1 | 95 | 4 |
| 1 | 100 | 2 | 94 | 4 |
| 4 | 100 | 8 | 88 | 4 |
| 8 | 100 | 16 | 80 | 4 |
| Blank | 100 | 0 | 100 | 0 |

Using a 100 mM IPTG stock and 50 g/L L-arabinose.
BL21(DE3).pU01.pU13 + L-arabinose (0.1 % (w/v)), varying IPTG (mM).

| [IPTG] (mM) | E. coli (μL) | v(IPTG) (μL) | v(MQ) (μL) | Arabinose (μL) |
| --- | --- | --- | --- | --- |
| 0 | 100 | 0 | 96 | 4 |
| 0.5 | 100 | 1 | 95 | 4 |
| 1 | 100 | 2 | 94 | 4 |
| 4 | 100 | 8 | 88 | 4 |
| 8 | 100 | 16 | 80 | 4 |
| Blank | 100 | 0 | 100 | 0 |

### Week 16

This final week of laboratory work we did our final fluorescence test. We tested our constructs with PFOA-sensitive aptamers and our strain BL21(DE3).pU03.pU07. Thus, following overnight cultures were prepared:

* BL21(DE3).pU01.pU11
* BL21(DE3).pU01.pU12
* BL21(DE3).pU01.pU13
* BL21(DE3).pU01.pU14
* BL21(DE3).pU03.pU07

For controls, overnight cultures of pIDT2 and pU02 were also prepared. All overnight cultures were prepared in LB with appropriate antibiotics. That being, kanamycin for pIDT2, chloramphenicol for pU02, and ampicillin and chloramphenicol for the remaining.

The following day, all strains were reinoculated to OD 0.05.

Stock solutions used for the inductions:

* aTc (5 μM)
* MnCl2 (20 mM, 2 μM, 200 μM)(tetrahydrate 197.91 g/mol)
* Theophylline (25 mM)
* L-arabinose (50 g/L)
* IPTG (100 mM)
* PFOA (0.02 mM and 2 mM)

| 0 | 0.01 | 0.1 | 0.25 | 0.5 | 1 | IPTG | L-ara | Blank |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0 | 0.01 | 0.1 | 0.25 | 0.5 | 1 | IPTG | L-ara | Blank |  |  |
| 0 | 0.01 | 0.1 | 0.25 | 0.5 | 1 | IPTG | L-ara | Blank |  |  |
| 0 | 0.01 | 0.1 | 0.25 | 0.5 | 1 | IPTG | L-ara | Blank |  |  |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank |  |  |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank |  |  |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | LB | MQ |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | pIDT2 Cryo | pU01 |

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | 0 | 0 | 0 |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | 0.1 | 0.1 | 0.1 |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | 1 | 1 | 1 |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | 10 | 10 | 10 |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | 100 | 100 | 100 |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | Blank | Blank | Blank |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | LB | MQ |  |
| 0 | 0.0001 | 0.001 | 0.01 | 0.1 | 0.9 | IPTG | L-ara | Blank | pIDT2 Cryo | pU01 | pIDT2 Cryo |

Column 6 contains PFOA dissolved in dPBS, not MQ.

Controls shown in blue are:

* LB: 200 μL LB
* MQ: 200 μL MQ
* BL21(DE3): 100 μL MQ + 100 μL BL21(DE3)
* pIDT2 cryo: 96 μL MQ + 100 μL pIDT2 from cryostock (showed growth) + 4 μL L-arabinose (0.1% w/v)
* pU01: 96 μL MQ + 100 μL pU01 (showed growth) + 4 μL L-arabinose (0.1% w/v)

Using a 100 mM IPTG, 50 g/L L-arabinose, 5 μM aTc (In MQ).
BL21(DE3).pU03.pU07 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying aTc (mM).

| [aTc] (μM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | aTc (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.01 | 100 | 4 | 93.6 | 2 | 0.4 |
| 0.1 | 100 | 4 | 90 | 2 | 4 |
| 0.25 | 100 | 4 | 84 | 2 | 10 |
| 0.5 | 100 | 4 | 74 | 2 | 20 |
| 1 | 100 | 4 | 54 | 2 | 40 |
| IPTG | 100 | 0 | 98 | 2 | 0 |
| L-ara | 100 | 4 | 96 | 0 | 0 |
| Blank | 100 | 0 | 100 | 0 | 0 |

Using a 100 mM IPTG, 50 g/L L-arabinose, (in orange) 2 mM PFOA (in MQ) and (in green) 0.02 mM PFOA (in MQ).
BL21(DE3).pU01.pU13 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying PFOA (mM).

| [PFOA] (mM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | PFOA (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.0001 | 100 | 4 | 93 | 2 | 1 |
| 0.001 | 100 | 4 | 84 | 2 | 10 |
| 0.01 | 100 | 4 | 93 | 2 | 1 |
| 0.1 | 100 | 4 | 84 | 2 | 10 |
| 0.9 | 100 | 4 | 4 | 2 | 90 |
| IPTG | 100 | 0 | 98 | 2 | 0 |
| L-ara | 100 | 4 | 96 | 0 | 0 |
| Blank | 100 | 0 | 100 | 0 | 0 |

Using a 100 mM IPTG, 50 g/L L-arabinose, (in orange) 2 mM PFOA (in MQ) and (in green) 0.02 mM PFOA (in MQ).
BL21(DE3).pU01.pU11 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying PFOA (mM).

| [PFOA] (mM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | PFOA (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.0001 | 100 | 4 | 93 | 2 | 1 |
| 0.001 | 100 | 4 | 84 | 2 | 10 |
| 0.01 | 100 | 4 | 93 | 2 | 1 |
| 0.1 | 100 | 4 | 84 | 2 | 10 |
| 0.9 | 100 | 4 | 4 | 2 | 90 |
| IPTG | 100 | 0 | 98 | 2 | 0 |
| L-ara | 100 | 4 | 96 | 0 | 0 |
| Blank | 100 | 0 | 100 | 0 | 0 |

Using a 100 mM IPTG, 50 g/L L-arabinose, (in orange) 2 mM PFOA (in MQ) and (in green) 0.02 mM PFOA (in MQ).
BL21(DE3).pU01.pU12 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying PFOA (mM).

| [PFOA] (mM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | PFOA (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.0001 | 100 | 4 | 93 | 2 | 1 |
| 0.001 | 100 | 4 | 84 | 2 | 10 |
| 0.01 | 100 | 4 | 93 | 2 | 1 |
| 0.1 | 100 | 4 | 84 | 2 | 10 |
| 0.9 | 100 | 4 | 4 | 2 | 90 |
| IPTG | 100 | 0 | 98 | 2 | 0 |
| L-ara | 100 | 4 | 96 | 0 | 0 |
| Blank | 100 | 0 | 100 | 0 | 0 |

BL21(DE3).pU01.pU014 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying manganese (μM).

| [Mn] (μM) | E. coli (μL) | v(arabinose) (μL) | v(MQ) (μL) | IPTG (μL) | Mn (μL) |
| --- | --- | --- | --- | --- | --- |
| 0 | 100 | 4 | 94 | 2 | 0 |
| 0.1 | 100 | 4 | 84 | 2 | 10 |
| 1 | 100 | 4 | 93 | 2 | 1 |
| 10 | 100 | 4 | 84 | 2 | 10 |
| 100 | 100 | 4 | 93 | 2 | 1 |
| 1000 | 100 | 4 | 84 | 2 | 10 |
| Blank | 100 | 0 | 100 | 0 | 0 |
| Mn concentrations (μM) | 2 | 200 | 20000 |  |  |
| [IPTG] (mM) | 1 | [arabinose] (%w/v) | 0.1 |  |  |

100 μL of each were transferred to a see-through microtiterplate and OD600 was measured. OD was not high enough after 5 h, so the plates were set to overnight incubation (13 h) at 37C. The fluorescence was measured the day after.

## Protocols

### Media

LB media was made using LB Broth (Lennox) from Sigma-Aldrich. 1 L broth was prepared by dissolving 20 g of LB Broth (Lennox) in 1 L of distilled water, and thereafter autoclaving the mixture. The finished media was kept at room temperature.

LB media with agar was using LB Broth with agar (Miller) from Sigma-Aldrich. 1 L was prepared by dissolving 40 g of LB Broth with agar (Miller) in 1 L of distilled water. The mixture was then autoclaved and put into a 60°C cabinet.

For liquid media or plates supplemented with antibiotics, 1000x dilutions were added to the media. This gave final concentrations of ampicillin (100 μg/mL), chloramphenicol (34 μg/mL), tetracycline (10 μg/mL), and kanamycin (50 μg/mL).

### Chemical competent cells

Adaption of protocols provided by R. Palm and J. Mejlsted.

A 3 day protocol for generating roughly 80x 50 ul aliquots for transformation protocols.

Materials:
Buffers

* 0.1 M CaCl2 (5.55 g in 500 mL)
* 0.1 M CaCl2/10% glycerol (in 50 mL 0.1 M CaCl2 add 500 uL glycerol 100%)

Media

* LB media
* LB agar plate

Other components

* An aliquot of competent *E. coli* cells
* Sterile 0.5 mL microtubes
* Sterile Erlenmeyer flasks
* Falcon tubes

Procedure:

Day 1

1. Around 4PM: Plate roughly 20 uL of competent cells on a LB agar plate without antibiotics.
2. Incubate plate at 37°C between 16-20 hours.

Day 2

3. At around 10 AM: Pick a single colony from the plate, and let it grow in 100 mL LB media at 37°C and 200 rpm for 6 to 8 hours.
4. At around 6 PM: Add 250 mL LB to a sterile Erlenmeyer flask. Add around 25 uL to 1 mL of culture to the media.
5. Let the culture grow overnight at 37°C.
6. Place buffers in the fridge.
7. Place pipettes, falcon tubes, and sterile 0.5 mL microtubes into the freezer.

Day 3

8. Measure OD600 of the overnight culture, and inoculate a Erlenmeyer flask with a volume so the final OD600 value in the culture becomes 0.01.
9. Grow the culture at 37°C with shaking, and measure OD regularly.
10. When an OD between 0.3-0.55 is reached, split up culture into 10x 50 mL falcon tubes (Each of them will have 25 mL of culture).

**From here on it is important that the cells, buffer and equipment remain at low temperature.**

11. Centrifuge the cells at 4000 rcf for 10 min at 4°C.
12. Add to each falcon tube 10 mL ice-cold 0.1 M CaCl2 and resuspend pellet by shaking the Falcon tubes (if possible avoid vortexing and repipetting).
13. Centrifuge the cells at 4000 rcf for 10 min at 4°C.
14. Resuspend pellet in 400 uL 0.1 M CaCl2/10%glycerol (vortex to resuspend the pellet).
15. Dispense 50 uL aliquots of suspension into 0.5 mL microtubes.
16. Store competent E. coli cells in -80°C.

### X7 PCR

Materials:

* rCutSmart buffer
* dNTPs (2 mM)
* Sterile MQ
* Primers (10 pmol/uL)
* X7 polymerase (2-3 U)
* DNA template (plasmid: 10 pg/uL)
* PCR tubes
* Thermocycler
* DMSO (optional)

Procedure:

1. Mix following in PCR tubes (adjust “# of reactions” if needed):

   | Reagent | Volume per reaction (uL) | Mastermix per volume (uL) |
   | --- | --- | --- |
   | rCutsmart | 10 | 20 |
   | dNTPs (2 mM) | 5 | 10 |
   | MQ | 31.5 | 63 |
   | Primer 1 (10 pmol/uL) | 1 | 2 |
   | Primer 2 (10 pmol/uL) | 1 | 2 |
   | X7 enzyme | 0.5 | 1 |
   | Template | 1 | - |
   |  | # of reactions | Total volume |
   |  | 2 | 98 |
2. Run the following program in a thermocycler (adjust elongation time to fragment size):

   | Step | Temperature (°C) | Time | Cycles |
   | --- | --- | --- | --- |
   | Activation | 98 | 30 s |  |
   | Denaturation | 98 | 10 s | x35 |
   | Annealing | 68 -0.5/cycles | 30 s | x35 |
   | Elongation | 72 | 1 min/kb | x35 |
   | End elongation | 72 | 10 min |  |
   | Hold | 12 | Infinite |  |
3. DpnI digestion of the template backbone (**Not needed if you do gel-band purification**):
   Add 1 µL DpnI to the clean PCR product.
   Incubate 30 min at 37°C (or 1 hr if the template is gDNA).
   Heat inactivate 20 min at 80°C.
4. Run a gel of 3 µL PCR product with dye to check for the correct band lengths.
   If there is only the correct band: PCR clean up.
   If there are multiple bands: run gel with remaining PCR product, excise, and gel purify.
5. Measure concentration of the fresh fragment using a NanoDrop.

### Plasmid transformation of *E. coli*

According to protocol by [GoldBio](https://goldbio.com/documents/4066/DH5-alpha%20Chemically%20Competent%20E.%20coli%20cells%20Transformation%20Protocol.pdf)

Materials:

* Competent *E. coli*
* LB with appropriate antibiotics
* 1-5 mL tubes
* Ice
* SOC media (recovery media, optional), otherwise LB
* Thermoshaker

Procedure:

1. Remove competent cells from -80°C freezer and thaw completely on ice (10-15 min).
2. Aliquot 1-5 µl (1 pg-100 ng) of DNA to the chilled microcentrifuge tubes on ice.
3. When the cells are thawed, add 50 µl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. Mix well by tapping. Do not pipette up and down or vortex to mix, this can harm cells and decrease transformation efficiency.
4. Incubate the cells with DNA on ice for 15 minutes. (BL21: 30 min).
5. After the 15-minute ice incubation, heat shock the cells at 42°C for 45 seconds. (BL21: 10 s).
6. Transfer the tubes to ice for 2 minutes. (BL21: 5 min).
7. SOC media (optional, not needed for ampicillin marker).

   Add 950 µl of Recovery Medium or any other medium of choice to each tube.

   Incubate tubes at 37°C for 1 hour at 210 rpm in a shaker incubator.
8. Spread 50 µl to 200 µl from each transformation on prewarmed selection plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies.
9. Incubate the plates overnight at 37°C.

### USER cloning

Materials:

* BioBLock DNA with USER overhangs
* USER-compatible opened vector
* CutSmart buffer
* Sterile MQ
* USER Enzyme Mix
* Competent *E. coli* (DH5α)
* Ice
* LB plates supplemented with appropriate antibiotics
* PCR tubes

Before conducting the cloning:

Keep total volume of mix at 10 μL (or alternatively 20 μL).

Add equimolar amounts of each fragment.

Procedure:
USER reaction mix

1. Mix following in a PCR tube:

   | Reagent | Volume (μL) | Negative control (μL) |
   | --- | --- | --- |
   | BioBlock DNA | 1-6 | 0 |
   | USER-ready vector | 1-6 | 1-6 |
   | CutSmart buffer | 0.5 | 0.5 |
   | MQ (up to 10) | x | x |
   | USER Enzyme | 1 | 1 |

Incubate as follows:

2. 37°C for 25-35 min.
3. Room temperature for 15-25 min.
4. Thaw competent *E. coli* DH5α on ice, while they are thawing:
5. Continue incubation for app. 10 min.
6. Put on ice, do not mix, do not vortex.

Transformation:

7. Mix the reaction with 10-50 μL competent *E. coli* DH5α - Competent cells should be handled gently.
8. Mix the DNA and cells gently by stirring with a pipette tip.
   Remember to add a positive control, using an unopened plasmid (typically 1 pg - 100 ng).
9. Incubate on ice for 5-15 min
10. Heat shock at 42°C for 0:50 - 1:30 min
11. Incubate on ice for 5-15 min
12. (Not needed for ampicillin marker) Add 950 μl SOC/LB media and incubate for 1 h at 37°C 250 rpm
13. Plate and spread on selective medium (LB + Amp, Kan/Neo or Cam)

### Invitrogen™ PureLink™ Quick Plasmid Miniprep Kit

[Miniprep isolation protocol (centrifuge)](https://assets.fishersci.com/TFS-Assets/LSG/manuals/purelink_quick_plasmid_qrc.pdf)

Follow this procedure to purify plasmid DNA using a centrifuge. Use a microcentrifuge capable of
centrifuging at >12,000 × g. For processing a large number of samples simultaneously, see the “Miniprep
plasmid isolation protocol (vacuum)”.

* Perform all centrifugation steps at room temperature using a microcentrifuge.
* Optional: Preheat an aliquot of TE Buffer (TE) to 65–70°C for eluting DNA. Heating is optional for eluting 1–30 kb plasmid DNA but is recommended for eluting DNA >30 kb.
* Ensure the bag containing the PureLink™ Quick Spin Columns is closed tightly after each use.
* Caution: Buffers contain hazardous reagents. Use caution when handling buffers.

Steps:

1. Harvest
   Centrifuge 1–5 mL of the overnight LB-culture. (Use 1–2 × 10^9 E. coli cells for each sample.) Remove all medium.
2. Resuspend
   Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous.
3. Lyse
   Add 250 μL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogeneous. Do not vortex. Incubate the tube at room temperature for 5 minutes.
4. Precipitate
   Add 350 μL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 × g for 10 minutes.
5. Bind
   Load the supernatant from step 4 onto a spin column in a 2 mL wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the flowthrough and place the column back into the wash tube.
6. Wash (Optional)
   (Recommended for endA+ strains). Add 500 μL Wash Buffer (W10) with ethanol to the column. Incubate the column for 1 minute at room temperature. Centrifuge the column at 12,000 × g for 1 minute. Discard the flowthrough and place column back into the wash tube.
7. Wash and remove ethanol
   Add 700 μL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 × g for 1 minute. Discard the flowthrough and place the column into the wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the wash tube with the flowthrough.
8. Elute
   Place the Spin Column in a clean 1.5 mL elution tube. Add 75 μL of preheated TE Buffer (TE) to the center of the column. Incubate the column for 1 minute at room temperature.
9. Recover
   Centrifuge the column at 12,000 × g for 2 minutes. The elution tube contains the purified plasmid DNA. Discard the column. Store plasmid DNA at 4°C (short term) or store the DNA in aliquots at −20°C (long term).

### Monarch® Plasmid Miniprep Kit from NEB

[Monarch® Plasmid Miniprep Kit from NEB](https://www.neb.com/en/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010)

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).
If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve.

Store Plasmid Neutralization Buffer (B3) at 4°C after opening, as it contains RNase A.

Note: unlike other commercial kits, all wash steps are required.

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.

*Note: For a standard miniprep to prepare DNA for restriction digestion or PCR, we recommend 1.5 ml of culture, as this is sufficient for most applications. Ensure cultures are not overgrown (12-16 hours is ideal).*

2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.

*Note: Care should be taken not to handle the sample roughly and risk shearing chromosomal DNA, which will co-purify as a contaminant. Avoid incubating longer than one minute to prevent irreversible plasmid denaturation.*

4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.

*Note: Be careful not to shear chromosomal DNA by vortexing or vigorous shaking. Firmly inverting the tube promotes good mixing, important for full neutralization.*

5. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.

*Note: Spin time should not be less than 2 minutes. Careful handling of the tube will ensure no debris is transferred and the 2 minute recommended spin can be successfully employed to save valuable time. For culture volumes > 1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Also, longer spin times will result in a more compact pellet that lower the risk of clogging the column.

To save time, spin for two minutes only.*

6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.

*To save time, spin for 30 seconds, instead of 1 minute.

If using a vacuum manifold instead of centrifugation, insert the column into a manifold and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.*

7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.

*Note: The collection tube is designed to hold 800 μL of flow-through fluid and still allow the tip of the column to be safely above the top of the liquid. Empty the tube whenever necessary to ensure the column tip and flow-through do not make contact.

To save time, spin for 30 seconds, instead of 1 minute.

If using a vacuum manifold, add 200 μL of Plasmid Wash Buffer 1 and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.
Make sure to follow the manifold manufacturer’s instructions to set-up the manifold and connect it properly to a vacuum source.*

8. Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for 1 minute.

*When using a manifold add 400 μL of Plasmid Wash Buffer 2 and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.*

9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.

*If using a vacuum manifold: Since vacuum set-ups can vary, a 1 minute centrifugation is recommended prior to elution to ensure that no traces of salt and ethanol are carried over to the next step.*

10. Add ≥ 30 μL DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

*Note: Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Delivery of the Monarch DNA Elution Buffer should be made directly to the center of the column to ensure the matrix is completely covered for maximal efficiency of elution. Additionally, yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated as a result of dilution. For larger plasmids (≥ 10 kb), heating the DNA Elution Buffer to 50°C prior to eluting and extending the incubation time after buffer addition to 5 minutes can improve yield.*

### Protocol for Ampliqon PureIT ExoZAP PCR CleanUp

[Protocol for Ampliqon PureIT ExoZAP PCR CleanUp](https://ampliqon.com/en/pcr-enzymes/pcr-enzymes/pcr-clean-up/pureit-exozap-pcr-cleanup/)

This protocol serves as a guideline for clean-up of 5 μl PCR product using PureIT ExoZAP PCR CleanUp\*.

Take out PureIT ExoZAP PCR CleanUp from the -20 °C freezer.

* Keep PureIT ExoZAP PCR CleanUp on ice at all times.
* Add 2 μl PureIT ExoZAP PCR CleanUp\*\* to 5 μl of amplified PCR product.
* Mix well and spin down.
* Incubate the reaction at 37 °C for 2-5 minutes to degrade remaining primers, single-stranded DNA and to inactivate excess nucleotides by dephosphorylation.
* Incubate at 80 °C to completely inactivate PureIT ExoZAP PCR CleanUp for 3-10 minutes.
* The cleaned up PCR product can now be used for downstream applications such DNA sequencing, primer extension experiment or SNP analysis.
* After treatment the PCR products can be stored at -20 °C

\*If treating PCR product of higher volume, then increase proportionally the amount of PureIT ExoZAP PCR CleanUp.

\*\*PureIT ExoZAP PCR CleanUp works in PCR buffers.

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

# Results

Photo: DTU

##### Menu

* [> Introduction](#introduction)
* [> Validation of the TMS (GFP)](#validation-of-the-tmsgfp)
* [> Validation of the new TMSs](#validation-of-new-tmss)
* [> Validation of PFOA TMSs](#validation-of-pfoa-tmss)
* [> Discussion and future perspectives](#discussion-and-future-perspectives)

## Introduction

The original tRNA-Mimicking Sequence (TMS) comes from the work conducted by Paul *et al.* (2020). Here, they achieve ligand-selective protein translation-inhibition by creating a tRNA-like structure modified by insertion of an aptamer sequence. Across our project, we tried to reproduce part of the work by Paul *et al.* (2020), and we expanded the TMS family with several new structures to be used as biobricks applicable mainly for biosensor production, and specifically for PFOA detection in water.

## Validation of the TMS (GFP)

The first step of our project was the reproduction of the TMS (GFP) from Paul *et al.* (2020), [BBa\_K4811002](http://parts.igem.org/Part%3ABBa_K4811002). This was tested as the composite part [BBa\_K4811018](http://parts.igem.org/Part%3ABBa_K4811018) in *E. coli* BL21(DE3), where the TMS is induced by addition of Isopropyl β-d-1-thiogalactopyranoside (IPTG). This construct was USER cloned into the high copy number pUC19 backbone for testing (see [Design](https://2023.igem.wiki/dtu-denmark/design) and [Engineering](https://2023.igem.wiki/dtu-denmark/engineering)).
A reporter, consisting of [BBa\_K4811005](http://parts.igem.org/Part%3ABBa_K4811005) was USER cloned into the low copy number pACYC184 backbone. This has GFP under the control of the P~~tet~~ promoter, and therefore is induced by anhydrotetracycline (aTc). It also has mCherry, under the control of the pBAD promoter, meaning L-arabinose will induce mCherry transcription. The mCherry transcript has the RBS [BBa\_K4811000](http://parts.igem.org/Part%3ABBa_K4811000) incorporated, meaning that translation of mCherry should be inhibited by the TMS (GFP). With a GFP aptamer incorporated in the D-loop, Paul *et al.* (2020) showed GFP interfering with this inhibition. Higher levels of GFP lead to more mCherry fluorescence.

A schematic of the system to be tested can be seen below:

![](https://static.igem.wiki/teams/4811/wiki/design/pu03-07-mrna.png)

GFP is induced by aTc, mCherry is induced by L-ara, and the TMS is induced by IPTG. The hypothesis is that the TMS is able to repress translation of mCherry, by binding to the RBS, and that GFP is able to bind to the TMS, thus, releasing the TMS from the mCherry RBS, allowing translation to begin.

First, the system was tested by induction with varying levels of L-arabinose and IPTG, to find the optimal induction conditions. The results of this can be seen below:

![Results_TMS(GFP), mCherry, IPTG](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/gfp-tms-varying-iptg.png)

mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of IPTG concentration. All measurements were with 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with no inducers present (uninduced)

![Results_TMS(GFP), mCherry, Arabinose](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/gfp-tms-varying-arabinose.png)

mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of L-arabinose concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG (IPTG), one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced)

As described in [Engineering](https://2023.igem.wiki/dtu-denmark/engineering), 1 mM IPTG and 0.1 % w/v L-arabinose were found to be sufficient conditions for simultaneous expression of mCherry and the TMS. Using these conditions, GFP was induced by aTc, which showed the following results:

![Results_TMS(GFP), GFP](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/gfp-tms-gfp-varying-atc.png)

GFP fluorescence normalized relative to OD, 488 nm excitation/545 nm emission, as a function of anhydrotetracycline (aTc) concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM IPTG, one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced)

The results showed that aTc induced GFP, with 0.5 μM having the highest fluorescence. We would have expected 1 μM GFP to have even higher fluorescence, however it seems to be lower, but with a much higher standard deviation associated with the data.
With respect to mCherry fluorescence, we were not able to recreate the results of Paul *et al.* (2020), and the only statistical significance was for the measurement of 1 μM of aTc, which had higher fluorescence than the other measurements. However, the corresponding GFP fluorescence was lower for 1 μM aTc than for 0.5 μM aTc. This may be an experimental error associated with the 1 μM aTc measurement. There is also no statistical significance between the 0 μM aTc measurement and the L-ara control, with only L-ara as inducer, and no IPTG.

![Results_TMS(GFP), mCherry](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/gfp-tms-mcherry-varying-atc.png)

mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of anhydrotetracycline (aTc) concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose, except the controls. As controls there is a culture induced with only 1 mM (IPTG), one only induced by L-arabinose (L-ara), and one with no inducers present (uninduced)

## Validation of new TMSs

Using existing aptamers from the literature, new TMSs were constructed. This includes two manganese responsive TMSs and two theophylline responsive TMSs. They were characterized as the following composite parts:

* [BBa\_K4811021](http://parts.igem.org/Part%3ABBa_K4811021)(PT7-TMS(Mn1)-TT7)
* [BBa\_K4811031](http://parts.igem.org/Part%3ABBa_K4811031) (PT7-TMS(Mn2)-TT7)
* [BBa\_K4811022](http://parts.igem.org/Part%3ABBa_K4811022)(PT7-TMS(Theo1)-TT7)
* [BBa\_K4811026](http://parts.igem.org/Part%3ABBa_K4811026)(PT7-TMS(Theo2)-TT7)

All TMSs were characterized in *E. coli* BL21(DE3), where the TMS is induced by IPTG through the T7 polymerase of the DE3 system. The constructs were USER cloned into the high copy number pUC19 backbone for testing.
A reporter, consisting of [BBa\_K4811003](http://parts.igem.org/Part%3ABBa_K4811003) was USER cloned into the low copy number pACYC184 backbone. This has mCherry under control of the pBAD promoter, meaning L-arabinose will induce mCherry transcription. The mCherry transcript has the RBS [BBa\_K4811000](http://parts.igem.org/Part%3ABBa_K4811000) incorporated, meaning that translation of mCherry should be inhibited by the TMS, if the inhibiting capabilities are conserved upon changing the D-loop.

A general schematic of the system to be tested can be seen below:

![](https://static.igem.wiki/teams/4811/wiki/design/pu01-0xai.png)

mCherry is induced by L-ara, and the TMS is induced by IPTG. The hypothesis is, that the TMS is able to repress translation of mCherry, by binding to the RBS, and that the ligand is able to bind to the TMS aptamer in the D-loop, releasing the mCherry RBS of the TMS, allowing translation to begin.

The two theophylline TMSs were designed using the paper published by Suess *et al.* (2004) for the aptamer sequence, and these were tested for mCherry fluorescence based on concentration of the compound theophylline. The results showed a statistically significant decrease in mCherry fluorescence dependent on theophylline concentration for TMS(theo2), but no statistical significance for TMS(theo1), which had a much higher standard deviation associated with its results. Together, this shows that it is possible to insert an aptamer sequence from literature and, with the appropriate design changes, adapt it to the TMS platform in order to create a TMS system that is responsive to the desired ligand in a dose-controllable way. Results are summarized below.

![Results_TMS(Theo_combined)](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/theo-tmss-combined.png)

mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of theophylline concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose.

Additionally, two manganese TMSs were tested, and the results are summarized in the figure below. A decrease in mCherry fluorescence can be seen in TMS(Mn1). To improve the Mn-dependent reporter translation inhibition, the principles that were successful in the design of TMS(Theo2) were applied to TMS(Mn2). This construct, however, had a less significant decrease in fluorescence than the first iteration of the engineering cycle.

![Results_TMS(Mn_combined)](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/mn-tms-combined.png)

mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of manganese concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose.

## Validation of PFOA TMSs

Applying the principles learned from the design of the above TMSs, 3 different PFOA aptamer designs were incorporated into the D-loop of the TMS structure starting from the aptamer described in Park *et al.* (2022), creating [TMS(PFOA1) - BBa\_K4811024](http://parts.igem.org/Part%3ABBa_K4811024), [TMS(PFOA2) - BBa\_K4811030](http://parts.igem.org/Part%3ABBa_K4811030), and [TMS(PFOA3) - BBa\_K4811025](http://parts.igem.org/Part%3ABBa_K4811025). These were tested in quadruplicates with varying concentration of PFOA and the results are summarized in the graph below.
All PFOA TMSs showed a decrease in mCherry fluorescence based on PFOA concentration. The best TMS designed is the one produced in the second iteration of the engineering cycle. Crucially, and perhaps interestingly, the original inserted aptamer sequence from the Park *et al.* (2022), selected through a PCR-dependent technique such as SELEX, is a DNA sequence, but our TMS(PFOA)s seem to work despite being in RNA form.

![Results_TMS(PFOA_combined)](https://static.igem.wiki/teams/4811/wiki/data/finalgraphs/pfoa-tmss-combined.png)

mCherry fluorescence normalized relative to OD, 561 nm excitation/617 nm emission, as a function of PFOA concentration. All measurements were with 1 mM IPTG and 0.1 % w/v L-arabinose

## Discussion and future perspectives

As mentioned in [Engineering](https://2023.igem.wiki/dtu-denmark/engineering), although contrary to what was expected from the original TMS paper, all the TMS biobricks produced in this work show a ligand-dependent inhibition of protein translation. This suggests a need to revise the mechanistic model of how the TMS structures exert their function. Overall, the produced TMSs show a concentration-dependent inhibitory activity whose specificity is conferred by the inserted aptamer sequence. Assuming that the observed correlation between PFOA and fluorescence is based upon a change in the structure of the TMS, a stabilization of the most stable inhibitory structure would explain the observed results.

As represented in the animation above, a ligand molecule binds to the D-loop of the TMS, and together the complex binds to the reporter mRNA. In this example the ligand represented is the theophylline molecule that binds to the TMS (Theo2) [BBa\_K4811011](http://parts.igem.org/Part%3ABBa_K4811011). Contrarily to the original TMS mechanism model, the presence of the ligand during translation leads to a reduction of fluorescence output, represented here as a dimming on the red shine. In regards to the main track of our project, represented in the diagram below, the ligand is PFOA binding to TMS (PFOA2) [BBa\_K4811030](http://parts.igem.org/Part%3ABBa_K4811030), and its presence stabilizes TMS binding on the mRNA RBS ([BBa\_K4811000](http://parts.igem.org/Part%3ABBa_K4811000), colored orange).

![Revised model](https://static.igem.wiki/teams/4811/wiki/images/mcherry-graph.svg)

Although the creation of various ligand-responsive TMSs was generally successful, it is worth noting that these behave in contradiction compared to what was initially expected. This is when only taken the work by Paul *et al.* (2020) into account. Thus, this might signal that reproducibility is dependent on other factors, which might have had an influence on our experimental. setup. Some variables that could have had an influence are pH, solubilized ions, and change in surface tension.

**Theophylline is a weak base**, so both the medium and intracellular pH might exert a slight effect across the theophylline experiments. The increase in effectiveness of the second version of the TMS compared to the first might shed light on the role of the structural features to look for during the part design. The original aptamer sequence selected contained a stem that connects the aptamer RNA sequence to the translated mRNA. This stem has the functional role of transmitting the conformational change of the aptamer upon theophylline binding to the mRNA by changing the accessibility of the RBS cassette through a one-nucleotide slipping mechanism. Although this mechanism exerts a response *in vivo* on the native mRNA, its function in a TMS design is unknown, and could make the TMS structure less sensitive to the ligand binding. Furthermore, its removal during the designing of TMS(Theo2) is seen to increase its sensitivity.

**Manganese is a solubilized ion**, which would change the osmotic pressure. One factor to be considered is that, the original aptamer inserted in the TMS structure was taken as a biobrick from the iGEM registry ([BBa\_K902074](http://parts.igem.org/Part%3ABBa_K902074)), and this sequence is native of *E. coli*, where it controls the production of the efflux pump for the export of manganese ions themselves. Hence, the intracellular concentration levels of manganese will appear broad on a population level, not only making *E. coli* not the best organism to characterize this structures into, but also likely leading to an organism-dependent effectiveness of the two Mn TMSs.

**PFOA changes the surface tension of water**, which may lead to better shaking of the culture during induction. Furthermore, association of PFOA to the bacterial cell membrane has been suggested in the literature but not deeply investigated, and possible disruption of cell membranes or membrane-associated proteins might impose additional stress conditions that should be accounted for while designing experiments.

All these factors could be explored to determine the mechanism of action of the TMS. Having said this, we managed to obtain a statistically significant, dose-dependant response for both theophylline TMSs, of which [BBa\_K4811011](http://parts.igem.org/Part%3ABBa_K4811011) shows the highest response, and two PFOA TMSs, with [BBa\_K4811030](http://parts.igem.org/Part%3ABBa_K4811030) showing the highest response. Naturally, further experimental confirmation is needed before definite conclusions about the produced TMSs can be reached. Specifically, a deeper insight into the mechanistic action of TMSs is necessary, and could aid the design of more TMSs in the future.
We therefore encourage future teams to work with the TMS system to elucidate the exact method of action, as there is potential for elucidation and we have seen a wide variablity between operators. For future experiments, the addition of rigorous negative controls might be advisable in order test the specificity of the system and to eliminate contributions of false positives. Further factors to be tested might include:

* Toxicity of the selected ligands
* Toxicity of reporter production-associated stress
* Expression of efflux pumps
* Culturing conditions
* Strain-dependency of the system
* Functionality in other types of organisms
* Effect of ligand-associated conformational change on TMS-mRNA interactions
* Re-design of the aptamer insertion area onto the TMS body

We strongly believe that with the contribution and validation of the scientific community, this technology can become a valuable tool for the designing of various biosensors, as well as a bioengineering tool for the study of gene function.

**You can find our raw data here:** [Raw data](https://static.igem.wiki/teams/4811/wiki/data/raw-data-combined.csv)

{% endblock %}