iGEM 2020 — Team UofUppsala

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# Team:UofUppsala/Notebook

# Uppsala iGEM 2020

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UofUppsala/Engineering

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* Project

  [Description](https://2020.igem.org/Team%3AUofUppsala/Description)
  [Design](https://2020.igem.org/Team%3AUofUppsala/Design)
  [Engineering success](https://2020.igem.org/Team%3AUofUppsala/Engineering)
  [Proof of Concept](https://2020.igem.org/Team%3AUofUppsala/Proof_Of_Concept)
* Lab

  [Wet lab](https://2020.igem.org/Team%3AUofUppsala/Results)
  [Dry lab](https://2020.igem.org/Team%3AUofUppsala/Model)
* [Parts](https://2020.igem.org/Team%3AUofUppsala/Parts)
* Future

  [Implementation](https://2020.igem.org/Team%3AUofUppsala/Implementation)
  [Contributions](https://2020.igem.org/Team%3AUofUppsala/Contribution)
* Human Practices

  [Integrated Human Practices](https://2020.igem.org/Team%3AUofUppsala/Human_Practices)
  [Education](https://2020.igem.org/Team%3AUofUppsala/Education)
  [Collaborations](https://2020.igem.org/Team%3AUofUppsala/Collaborations)
* [Safety](https://2020.igem.org/Team%3AUofUppsala/Safety)
* Team

  [Meet the Team](https://2020.igem.org/Team%3AUofUppsala/Team)
  [Attributions](https://2020.igem.org/Team%3AUofUppsala/Attributions)
* [Awards](https://2020.igem.org/Team%3AUofUppsala/Awards)

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# Engineering Success

#### Index

* [Development of iGEM Type IIS standard](#TypeIIS)
* [Prototype of NANOFLEX hardware](#Prototype)
* [References](#References)

**On this page, we show the aspects in which we succeed with the engineering cycle of : Research → Imagine → Design → Build → Test → Learn → Improve → Research.**

# Development of iGEM Type IIS Standard

---

Type IIS assembly methods allow the assembly of multiple DNA parts simultaneously, so it can easily generate many different transcriptional units. Type IIS restriction enzymes enable a one pot reaction due to the fact that they are offsite cutters: they cut the DNA at a specific distance from their recognition site. Therefore, specific overhangs or fusion sites can be designed for each DNA part, which leads to multiple DNA fragments being assembled in the correct order and orientation.

iGEM recently described a Type IIS assembly standard RFC[1000] which is based on Modular Cloning (MoClo) [(1)](#(1)) and Loop assembly [(2)](#(2)), and it makes use of SapI and BsaI enzymes. However, since this standard is fairly new, there is a lack of information and a need of improvement. In this section we describe our work in developing this standard.

## iGEM Type IIS guidebook

The general characteristics of this cloning method are presented in the  [iGEM Type IIS assembly page](http://parts.igem.org/Help%3AStandards/Assembly/Type_IIS). Other pages such as the Type IIS backbone registry pages (for example [pSB1C00](http://parts.igem.org/Part%3ApSB1C00)) and the MoClo [(1)](#(1)) and Loop [(2)](#(2)) papers contain supplementary information which is needed in order to make use of the method.

In order to make iGEM Type IIS assembly more accessible, we have compiled this information together with our experiences in design and lab work in a guidebook. We hope that this guidebook will encourage more teams to use the iGEM Type IIS standard, and that it is of assistance throughout the design and cloning process!

[iGEM Type IIS guide](https://static.igem.org/mediawiki/2020/e/e2/T--UofUppsala--typeIIS_guide_new.pdf)

The iGEM registry is an important source of information for both iGEM teams and the synthetic biology community alike. We therefore created a collection for iGEM Type IIS specific parts that are necessary or convenient when designing and cloning using this assembly method. You can read more about this collection in [our Parts page](https://2020.igem.org/Team%3AUofUppsala/Parts#Collection) and in the [registry](http://parts.igem.org/Collections/iGEM_Type_IIS_Collections).

## Dummy Parts

When we started working with the iGEM Type IIS standard, we realized the need to have dummy parts. Thus, we decided to create these parts since the beginning, to test and improve them through the course of our project.

##### Research, Imagine, Design

Type IIS iGEM standard allows for fast and efficient assembly of constructs in groups of four components. Sometimes, four components might not be needed in higher level assemblies.

Two possible solutions were considered. The existing parts might be modified so that they ligate and take place of two components. However, this strategy would require custom design and thus not lead to the creation of standard parts. The second option is to create a placeholder parts which have no function but can be included whenever they are needed. These placeholders are called dummy parts and were created based on an existing Modular Assembly method[(3)](#(3)).

##### Build

Two different dummies (TU-DY (BBa\_K3425017) and MTU-DY (BBa\_K3425018)) were ordered and cloned into all different pOdd and pEven plasmids. With this, parts which mimic all possible transcriptional units (pSB1K01-DY to pSB1K04-DY) and multi-transcriptional units (pSB3C11-DY to pSB3C14-DY) were assembled and sequenced.

##### Test, Learn

In order to check whether these would work as dummy parts, all four pSB1K0#-DY mimicking four TUs were cloned together into pSB3C11. Similarly all four dummies mimicking MTUs pSB3C1#-DY were cloned into pSB1K02. Sequencing revealed that only one out of six clones contains the expected sequence. The remaining five clones contain deletions or insertions of various sizes, all of them in the area of the “multi-dummy” sequence. These dummy parts are likely unstable when they are next to each other, since they generate a short and repetitive sequence. This might have caused polymerase slippage, a phenomenon that happens when there are reiterated sequences [(4)](#(4)).

##### Improve

A simple solution would be not cloning the dummy sequences next to each other. There might be a situation where this is not possible, so in that case it might be good to use longer dummy parts that are all different from each other. These parts were designed *in silico* by retrieving random 8bp DNA sequences from the Random DNA Generator [(5)](#(5)) and adding the corresponding fusion sites. They were added in the parts registry as pSB1K0#-DY (BBa\_K3425023 to 26) and pSB3C11 (BBa\_K3425027 to 30).

Further information and discussion about this topic together with other Type IIS related experiments can be found [here](https://2020.igem.org/Team%3AUofUppsala/Results#TypeIIS).

## New pOdd Backbones for Level 3 Assemblies

An important avenue of improvement for the iGEM Type IIS standard is the introduction of new pOdd backbones. The current pOdd backbones, pSB1K0#, are high copy number backbones which are suitable for Level 1 assemblies. The high copy number allows for higher yield from plasmid extraction, which makes it easier to obtain enough DNA for sequencing and assembling transcriptional units into Level 2 plasmids.

High copy number backbones, however, are less than ideal for higher level assemblies, and iGEM Headquarters advises against using them for this purpose (see  [pSB1K01](http://parts.igem.org/Part%3ApSB1K01)). The metabolic burden caused by the expression of multiple transcriptional units in a high copy number plasmid can be too overwhelming for the cell, which might make the plasmid unstable and prone to mutation or loss [(6)](#(6)).

For this reason, new sets of medium (10-12) and low (~5) copy number pOdd backbones were designed and assembled by our team. You can read more about these new backbones and other Type IIS related experiments [here.](https://2020.igem.org/Team%3AUofUppsala/Results#TypeIIS)

# Prototype of NANOFLEX Hardware

---

To realistically argue the possibility of our kit reaching the users we developed a prototype of the hardware that will contain our cellular biosensor. While sketching our prototypes and discussing prototyping and life cycle assessment with experts, we came to define 6 key concepts for the envisioned kit.

![](https://static.igem.org/mediawiki/2020/2/21/T--UofUppsala--final_prototype.png)

**Figure 1.** Our prototype
 Our final prototype taking into account the six concepts we determined while redesigning our ideas.

#### Key Concepts Dictating Kit Design

1. **Biological prerequisites for hardware of NANOFLEX kit:** separated chambers for bacteria, media and sample.
2. **Functionality of the hardware:**  flow between the chambers, locking mechanisms.
3. **User safety:** leakage prevention and seals.
4. **Manufacturing and materials:** with respect to environment and functionality.
5. **Intuitive design for the user:** clean design without exposure of small elements.
6. **Safe disposal and recycling:** designed for returning to the distributor.

### Development of the Prototype

Below you can read about our way towards a kit with examples of pitstop prototypes that influenced the final design the most.

#### Biological Prerequisites for Hardware of the NANOFLEX Kit

**1. Media chamber:** this chamber holds sterile media. In the case the reporter module of NANOFLEX is an enzyme, the media would also contain the respective substrate. For example, if beta-galactosidase is the reporter enzyme, this chamber would also hold (solubilized) X-gal.

**2. Bacteria chamber:** this chamber would have no media, containing only the lyophilized bacteria that we have engineered. When using this kit, the sterile media mentioned previously would first have to enter this chamber, in order to reactivate the bacteria and induce cell growth.

**3. Sample chamber:** this is the chamber in contact with the user. The sample (a blood sample or sputum smear diluted in water) is meant to be placed here. The design of the kit needs to ensure that our engineered bacteria, once reactivated, come into contact with the sample. The chambers need to be transparent as well, since the test results are to be seen by the naked eye.

![](https://static.igem.org/mediawiki/2020/8/8a/T--UofUppsala--prototype1.png)

**Figure 2.** Our first prototype

#### Our First Prototype

In **Figure 2** you can see our first prototype. In this early version of the kit, the sterile media is stored in the white chamber; the blue section contains x-gal and our engineered bacteria, and both chambers are separated by a membrane. The sample would be collected with a swab, which the user would then use to pierce the membranes separating the chambers. Once the reaction is finished, the user would pierce yet another membrane, allowing both our bacteria and the contents of the sample to be mixed with a neutralization solution, present in the last chamber (colored red in the figure). Issues found with this design: user safety was not ensured; unsure flow between chambers; possibility of leakage due to lack of integrity in a membrane-based design.

#### Functionality of the hardware

We discussed our prototyping with a prototyping consultant, Charlie Carpene. And were suggested to put extra thought in the following points.

**Ensuring flow between the chambers.** As the biological prerequisites require a three-chamber system, where all the contents combine at the end, we had to engineer with the flow between the chambers to be secured. Meaning, the combination of the chamber contents cannot be hindered by the initial sealing of each chamber.

**Preventing leakage.** The flow between the chambers needs to be controlled as well. No leakage should take place as the contents of the kit, e.g. genetically modified organisms need to be contained.

![](https://static.igem.org/mediawiki/2020/9/9d/T--UofUppsala--prototype2.png)

**Figure 3.** Our second prototype

### Our Second Prototype

This prototype has user safety in mind as the system would seal permanently upon combination of both chambers. The inner chamber is minimal and the outer scaffold would allow easier handling of the kit. However, we continued working on other possible designs, as the visual of this one is not as clean as we intended; more importantly, this prototype may also compromise the safety of the user, due to both the risk of leakage and the considerable exposure of the bacteria chamber prior to usage of the kit.

#### User Safety

Even though our modified organism does not impose any direct risk to the user or environment, the contents should not be possible to be in contact with no one else than the manufacturer. Therefore, we need locking mechanisms and a rigid scaffold. The individual chambers should be sealed separately initially and the locking mechanism upon combining the chamber contents need to seal the whole kit. While designing we looked into different locking mechanisms:

**Membranes:** these achieve effective sealing for many chemical/biological products. However, when designing the kit, it must be taken into consideration that the membranes should not be exposed to the user: direct contact with the membrane could compromise both the users safety and the correct functioning of the kit. See **Figure 5.**

**Hooks:** with inspiration from laboratory waste bins that seal permanently, we explored implementation of hooks. See **Figure 4**.

**Thread:** intuitive “screw together”-design can be achieved, see **Figure 4**. In order to make the system impossible to be unscrewed, the thread needs to be functional in one direction.

**Tesla valve:** a channel that due to fluid mechanics is impossible to flow backwards, see **Figure 6**.

![](https://static.igem.org/mediawiki/2020/9/90/T--UofUppsala--hook_and_thread.png)

**Figure 4.** Hook locking mechanism (above) and thread locking mechanism (below)

![](https://static.igem.org/mediawiki/2020/c/c1/T--UofUppsala--membranes.png)

**Figure 5.** Membranes in our final design, marked in yellow (below)

![](https://static.igem.org/mediawiki/2020/5/5e/T--UofUppsala--tesla.png)

**Figure 6.** Tesla valve

#### Manufacturing and Materials

From the conditions listed above we had to decide on the material for the kit, which in itself loops back to the design options allowed.

**Plastic scaffold.** The conditions above, movable parts and transparency, sets quite stringent criteria on the materials. We envision our kit to be made of hard plastic. While assessing the life cycle of an envisioned kit we began thinking of the environmental impact of our kit. The design must minimize the dimensions, using the optimal amount of plastic, see **Figure 7**. Shortly, the kit should be as compact as possible without being hard to handle and preferably reusable. Read more about the principles of Life Cycle Assessment [here.](https://2020.igem.org/Team%3AUofUppsala/Human_Practices#Kit)

**Aluminum membranes for separating chambers.** Incorporation of membranes means more intricate manufacturing and quality control process. There are more steps required in combining the different materials *i.e.* plastic scaffold and an aluminum membrane. As previously mentioned the membrane should not be directly reachable by the end-user, however, during manufacturing they placement of the membrane in the scaffold must be operative.

**Rubber/glue rims to prevent leakage.** An additional material that can be incorporated in the design is rubber or glue rims along the moving parts. This would prevent eventual leakage but contribute to a more intricate manufacturing as well.

![](https://static.igem.org/mediawiki/2020/f/f6/T--UofUppsala--measures.png)

**Figure 7.** Measures of our final system, mm

#### The Final Prototype

The final prototype takes into account biological prerequisites, manufacturer and end user. The prototype is made of two parts, sample holder and reaction chamber. In manufacturing the reaction chamber is separate allowing placing the membranes separating bacteria and media chambers. The membranes are deep in the structure increasing the safety. The sample holder serves as a hook locking mechanism that seals the kit permanently upon performing a test. The dimensions of the sample holder ensure piercing of the membranes and flow of the media into the bacteria chamber. The rounded bottom is to ensure easier observation as the bacteria will sediment with time.

[![

](https://static.igem.org/mediawiki/2020/2/21/T--UofUppsala--final_prototype.png)](https://static.igem.org/mediawiki/2020/7/74/T--UofUppsala--final_prototype_video.mp4)

After developing the prototype on paper we created the final design in Fusion 360 and Rhino oft. Then, with help of the 3D lab in Uppsala University, Uprint, we 3D printed a physical prototype of the NANOFLEX kit. See figure 8 below.

![3dprot](https://static.igem.org/mediawiki/2020/1/19/T--UofUppsala--prototype.png)

**Figure 8.** 3D-printed prototype of NANOFLEX.

#### Envisioned Distribution: Use and Disposal of our Kit

The kit would be distributed together with a sterile swab and an ampule with sterile water for applications where a swab sample. The water would be emptied in the sample chamber and thereafter, the sample transferred from swab to the chamber. For liquid samples a sterile, single use pipette would be distributed together with the kit.

After using the kit the system is permanently sealed and should be returned to the distributor. The recycling process for reusing the hardware for NANOFLEX tests depends on the manufacturers being able to reopen the system, sterilize it and replace the membranes without damaging the locking mechanism. Otherwise the plastic can be sterilized and recycled as other hard plastic waste.

# References

---

1. Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marillonnet, S. (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE. 6, e16765
2. Pollak, B., Cerda, A., Delmans, M., Álamos, S., Moyano, T., West, A., Gutiérrez, R. A., Patron, N. J., Federici, F., and Haseloff, J. (2019) Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytologist. 222, 628–640
3. Binder, A., Lambert, J., Morbitzer, R., Popp, C., Ott, T., Lahaye, T., and Parniske, M. (2014) A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants. PLOS ONE. 9, e88218
4. Levinson, G., and Gutman, G. A. (1987) Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol Biol Evol. 4, 203–221
5. Random DNA Generator [online] https://www.faculty.ucr.edu/~mmaduro/random.htm (Accessed October 16, 2020)
6. Jones, K. L., Kim, S.-W., and Keasling, J. D. (2000) Low-Copy Plasmids can Perform as Well as or Better Than High-Copy Plasmids for Metabolic Engineering of Bacteria. Metabolic Engineering. 2, 328–338
7. Chang, H. J., Mayonove, P., Zavala, A., De Visch, A., Minard, P., Cohen-Gonsaud, M., and Bonnet, J. (2018). A modular receptor platform to expand the sensing repertoire of bacteria. ACS synthetic biology, 7(1), 166-175.

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# Team:UofUppsala/Experiments

# Uppsala iGEM 2020

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UofUppsala/Results

Navigation Bar

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* Project

  [Description](https://2020.igem.org/Team%3AUofUppsala/Description)
  [Design](https://2020.igem.org/Team%3AUofUppsala/Design)
  [Engineering success](https://2020.igem.org/Team%3AUofUppsala/Engineering)
  [Proof of Concept](https://2020.igem.org/Team%3AUofUppsala/Proof_Of_Concept)
* Lab

  [Wet lab](https://2020.igem.org/Team%3AUofUppsala/Results)
  [Dry lab](https://2020.igem.org/Team%3AUofUppsala/Model)
* [Parts](https://2020.igem.org/Team%3AUofUppsala/Parts)
* Future

  [Implementation](https://2020.igem.org/Team%3AUofUppsala/Implementation)
  [Contributions](https://2020.igem.org/Team%3AUofUppsala/Contribution)
* Human Practices

  [Integrated Human Practices](https://2020.igem.org/Team%3AUofUppsala/Human_Practices)
  [Education](https://2020.igem.org/Team%3AUofUppsala/Education)
  [Collaborations](https://2020.igem.org/Team%3AUofUppsala/Collaborations)
* [Safety](https://2020.igem.org/Team%3AUofUppsala/Safety)
* Team

  [Meet the Team](https://2020.igem.org/Team%3AUofUppsala/Team)
  [Attributions](https://2020.igem.org/Team%3AUofUppsala/Attributions)
* [Awards](https://2020.igem.org/Team%3AUofUppsala/Awards)

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# Wet Lab

#### Index

* [Development of iGEM Type IIS standard](#TypeIIS)
  + [Mutagenesis of pSB3C0#](#Mutagenesis)
  + [New pOdd backbones](#NewBackbones)
  + [Creation of dummy parts](#Dummy)
* [First Generation](#FirstGen)
  + [DNA-binding domain](#DBD)
* [Next Generation](#NextGen)
  + [Detection Module with an Integrated Amplification Step](#Integ)
  + [Gram-positive host](#GramPositive)
  + [Signal transduction and amplification](#Amplification)
* [Protocols](#Protocols)
* [References](#References)

**On our wet lab page you will find information about the experiments that we performed in the lab. Furthermore, you will find our protocols and links to our notebooks. You can read more about the design behind it [here.](https://2020.igem.org/Team%3AUofUppsala/Design)**

# Development of iGEM Type IIS Standard

*Improvement of registered backbones, new Type IIS backbones and creation of placeholder parts applicable in multi-transcriptional unit design. Experiments in this section were performed to develop cloning methods to create NANOFLEX parts, but are not describing the implementation of NANOFLEX .*

![](https://static.igem.org/mediawiki/2020/9/97/T--UofUppsala--arrow-lab.svg)

---

Type IIS assembly was used for this project as it allows for easy and fast construction of many different transcriptional units. The flexibility of this cloning method is needed in order to tune the sensitivity of the biosensor since it allows us to comfortably modify the expression level of a certain coding sequence (CDS) by changing the promoter and RBS. This would allow the optimization of the expression level of all components which will help avoid false-positive results. Type IIS can also assemble the multiple transcriptional units that comprise the NANOFLEX system together in the same plasmid. However, iGEM Type IIS standard is fairly new, and is thus in need of improvement. In this section, we describe the experiments and new parts that were constructed for the sake of developing this standard. For more information about this assembly standard you can check the info in the [iGEM registry](http://parts.igem.org/Help%3AStandards/Assembly/Type_IIS) and for more extensive details read our [Type IIS guidebook](https://static.igem.org/mediawiki/2020/9/9c/T--UofUppsala--iGEM_Type_IIS_guidebook_Uppsala_2020.pdf).

## Mutagenesis of pSB3C0# pEven Loop Vectors

The sequence of pEven Loop vectors (for Level 2 assemblies) contains an additional, illegal BsaI site. BsaI is the enzyme which is used to cleave the assembly out of a pEven vector to be cloned back to a pOdd vector. Therefore, the presence of this illegal site would impact the construction of Level 3 assemblies.

On one hand, this additional site should not be a huge problem as it merely destroys the plasmid you are no longer interested in. On the other hand, it means that there is an additional site where the BsaI enzyme cuts, which could decrease the efficiency of the cloning reaction.

![Mutagenised Type IIS Level 2 backbones](https://static.igem.org/mediawiki/2020/9/9b/T--UofUppsala--20200814_backbones_fixed.jpg)

**Figure 1.** 1% Agarose Gel Electrophoresis (stained with SYBR Safe) analysis of BsaI digested plasmids. MW marker is GeneRuler 1kb Plus DNA Ladder (Thermofisher). pSB3C03 was linearised with EcoRI.

Therefore, Site-directed Mutagenesis by PCR was conducted on the pSB3C0# plasmids in order to remove the illegal sites. The new plasmids were named pSB3C1# and they can be found in our [Type IIS parts collection](http://parts.igem.org/Collections/iGEM_Type_IIS_Collections). Thus, pSB3C01 became pSB3C11 ([BBa\_K3425001](parts.igem.org/Part%3ABBa_K3425001)), pSB3C02 became pSB3C12 ([BBa\_K3425002](parts.igem.org/Part%3ABBa_K3425002)), and so on.

After PCR mutagenesis, screening was done by digestion of the plasmid DNA with BsaI (**Figure 1**). All backbones (pSB3C0# and pSB3C1#) contain a band that corresponds to the mRFP1 cassette ([BBa\_J04455](parts.igem.org/Part%3ABBa_J04455)) with a size of 1093bp. Then, pSB3C1# consist of one more band of 2725pb. Since pSB3C0# have one more BsaI site, they should present two more bands of 2434bp and 291bp. While the 291bp band is not visible, probably due to too low DNA amount, the shift in size before and after mutagenesis is clearly visible.

This difference is not visible, however, for pSB3C01 and pSB3C11. In addition, a band of longer DNA fragments can be seen for pSB3C01, but a comparison to well 10 shows that it corresponds to linearized (only single cut) plasmid, indicating that the digest was only partially complete. To investigate the lack of difference between pSB3C01 and pSB3C11, as well as to confirm the mutagenesis by another method, pSB3C01 and pSB3C1# were sequenced.

![Detail of the sequencing result of (A) pSB3C01 and (B) pSB3C11](https://static.igem.org/mediawiki/2020/0/0c/T--UofUppsala--pSB3C01_C11_mut.jpg)

**Figure 2.** Detail of the sequencing result of (A) pSB3C01 and (B) pSB3C11. At the upper part, the BsaI cutting site and mutagenesis primers are shown. At the bottom are the corresponding sequencing reads.

Sequencing results showed successful PCR mutagenesis for all of the pSB3C1# backbones, with the expected point mutation (**Figure 2B**). Furthermore, they also showed a point mutation in the original backbone, pSB3C01, (at a different point than the planned one) which eliminated the extra BsaI site (**Figure 2A**).

Afterwards, some assemblies into pSB3C1# were conducted. One example that was sequenced is the assembly of a dummy part into pSB3C11. You can read more about dummy parts [here](#Dummy), and find the sequencing results in the [Parts page](https://2020.igem.org/Team%3AUofUppsala/Parts). The results confirmed that these backbones would work as the new pEven set of vectors.

## New pOdd Backbones

One of the problems of iGEM Type IIS standard is the lack of pOdd backbones for Level 3 assemblies. While assembling from pSB3C1# to pSB1K0# is not a problem *in silico*, it is highly likely that it would not work *in vivo*. The reason for this is the high copy number of the currently available pOdd vectors (pUC19-derived pMB1 ori, 100-300 copies), as Level 3 assemblies contain multiple transcriptional units, which would make the plasmid toxic to the cells and thus unstable [(1)](#(1)).

Three sets of Level 3 plasmids have been designed in order to suit different situations. Each set comprises four plasmids which have the mRFP1 device for pOdd plasmids (BBa\_J04454) flanked by the corresponding Type IIS fusion sites substituting the BioBrick sites of the base plasmid. The first one, pSB3K0#, is derived from the pSB3K3 plasmid. These pOdd backbones have the same copy number as the existing pEven backbones (p15A pMR101-derived ori, 10-12 copies).

However, there are situations where this low to medium copy number is still too high. One such example is NANOFLEX, where including all the components would bring the plasmid length over 15kb. This would leave two possible strategies: genomic integration or a very low copy number plasmid (~5 copies). Genomic integration was the chosen strategy [when working with *B. subtilis*](#Bacillus), and two sets of very low copy number plasmids were created for *E. coli* constructs.

One set, pSB4K0#, is derived from the pSB4K5 (pSC101 ori) plasmid. These plasmids keep the alternating Cm-Kan resistances on Type IIS backbones. However, there might be a situation in which a very low copy number plasmid has to be co-transformed with a high copy number plasmid. For this reason, pSB4A0# were derived from the pSB4A5 plasmid (pSC101 ori). This last set can be co-transformed with any of the already existing backbones, allowing even triple transformation as an option for special cases.

Gibson assembly was used to construct these backbones. Afterwards, it was needed to screen for plasmids which contained the mRFP1 device and had the correct antibiotic resistance and copy number. Antibiotic resistance was screened by plating the transformation, the presence of the mRFP1 device by PCR and the copy number by sequencing the ori and by controlled plasmid extraction.

![1% Agarose Gel Electrophoresis analysis of (A) VF2-VR PCR and (B) full plasmid](https://static.igem.org/mediawiki/2020/d/d3/T--UofUppsala--20201001_gibson.png)

**Figure 3.** 1% Agarose Gel Electrophoresis (stained with Diamond Nucleic Acid Dye) analysis of (A) VF2-VR PCR and (B) full plasmid. MW marker is GeneRuler 1kb Plus DNA Ladder (Thermofisher).

The Gibson assembly did not yield too many colonies and there was no time to repeat it, so there were no samples of pSB3K03, pSB4K01 and pSB4K02. The mRFP1 device (1396bp amplification) was present in all available pSB3K0# and pSB4K02 (**Figure 3A**).

The full backbone amplification (3756bp for pSB3K0#, 3567bp for pSB4K0# and 4278bp for pSB4A0#) is positive for pSB4K04 and all pSB4A0# (**Figure 3B**). Although the band in pSB4A03 is barely visible amplification at the 4278bp length is observed, so it is considered as positive too. All these samples except pSB4A03 have multiple bands, which we believe are non-specific and are due to the long extension times required for full backbone amplification. Even with multiple bands, the strongest band corresponds to the expected size.

A smear can be seen in all samples (**Figure 3**), which we suspect is due to the use of Diamond Dye (Promega). The smear is present in samples which seem to have a lot of DNA and can also be seen in the MW marker. It is something we have consistently observed when using Diamond Dye.

It is concerning how in each gel half of the samples were negative, but it was different samples each time. Judging by the results (**Figures 3-6** and **8**), it seems to be an error in the handling of samples. It would be good to repeat it, but there was no time. From the available results, however, it seems that all backbones are correct.

To evaluate the copy number of these new backbones, two different experiments were conducted. First, sequencing of the origin of replication and then controlled plasmid DNA extraction.

A region crucial for copy number regulation, the origin of replication (ori), was sequenced (**Figures 4-6**).

![Sequencing of the origin of replication p15A of pSB3K02.](https://static.igem.org/mediawiki/2020/f/f2/T--UofUppsala--pSB3K02_ORI.png)

**Figure 4.** Sequencing of the origin of replication p15A of pSB3K02. The sequence is in two lines and the two sequencing reads correspond to two different clones.

In **Figure 4**, the sequencing results of pSB3K02 can be seen. It shows multiple single nucleotide changes in two different clones when comparing it to the expected sequence. It is unclear in which step the mutations were generated. Full length sequencing results are available in the [Parts page](https://2020.igem.org/Team%3AUofUppsala/Parts).

![Representative sequencing of the origin of replication pSC101 of pSB4A0#.](https://static.igem.org/mediawiki/2020/f/f6/T--UofUppsala--pSB4A0_1-4_ORI.png)

**Figure 5.** Representative sequencing of the origin of replication pSC101 of pSB4A0#. From top to bottom, the sequencing reads correspond to pSB4A01, pSB4A02, pSB4A03 and pSB4A04.

In **Figure 5**, the sequencing results of pSB4A01-4 can be seen. They show no mutations in the ori of all four backbones when comparing it to the expected sequence. Full length sequencing results for all backbones are available in the [Parts page](https://2020.igem.org/Team%3AUofUppsala/Parts).

![Representative sequencing of the origin of replication pSC101 of pSB4K02 and pSB4K04.](https://static.igem.org/mediawiki/2020/d/d7/T--UofUppsala--pSB4K0_2_4_ORI.png)

**Figure 6.** Representative sequencing of the origin of replication pSC101 of pSB4K02 and pSB4K04. From top to bottom, the sequencing reads correspond to pSB4K02 and pSB4K04.

![1% Agarose Gel Electrophoresis (stained with Diamond Nucleic Acid Dye) analysis of controlled minipreps (undigested)](https://static.igem.org/mediawiki/2020/5/57/T--UofUppsala--20200926_DNA_amount_light.jpg)

**Figure 7.** 1% Agarose Gel Electrophoresis (stained with Diamond Nucleic Acid Dye) analysis of controlled minipreps (undigested). MW marker is GeneRuler 1kb Plus DNA Ladder (Thermofisher).

In **Figure 6**, the sequencing results of pSB4K02 and pSB4K04 can be seen. They show no mutations in the ori when comparing it to the expected sequence. Full length sequencing results for both backbones are available in the [Parts page](https://2020.igem.org/Team%3AUofUppsala/Parts).

According to QIAGEN, a simple way to distinguish high and low copy number plasmids is to perform a miniprep [(2)](#(2)). Controlled plasmid extraction was conducted in order to see the difference between high (100-300), medium (15-20) and low (~5) copy number. This meant standardising plasmid DNA amounts to pellet weight that was used for its extraction, both the plasmids constructed by Gibson and controls for high (pSB1K01), medium (pSB3C11) and low (pSB4K5, pSMART [(3)](#(3))) copy number. The amount of DNA was measured in two different ways: via Nanodrop and via agarose gel electrophoresis.

Standardised plasmid amounts were loaded on an agarose gel (**Figure 7**). Strangely, the controls (except pSMART) have much higher amounts of DNA than the plasmids constructed by Gibson, in spite of both groups having medium and low copy number plasmids. This is also seen in standardised Nanodrop results (**Figure 8**), which also shows that the control for medium copy number has a higher amount of DNA than the control for high copy number. A box plot of the results (**Figure 9**) suggests no difference between medium and low copy number.

An ANOVA test was conducted, however, to see if there was any difference between groups (high, medium, low copy number). The results were non-significant (p=0.52). Even with the clear medium copy number outlier removed (pSB3C11), the test was non-significant (p=0.21). Even if the sample size was small, there is far too much overlap in the standardised values of different copy numbers. Moreover, there was a clear difference in RFP intensity between colonies with high and medium copy number plasmids in plates (Results not shown) when the control strains were used in other experiments. We can conclude that this method is not sensitive enough to distinguish between them, or that more replicates are needed.

![Level 3 backbones minipreps histogram](https://static.igem.org/mediawiki/2020/f/fc/T--UofUppsala--TypeIIS_histogram.png)

**Figure 8.** Histogram of the standardized amount of DNA (y) in each of the plasmid minipreps (x)

![Level 3 backbones minipreps boxplot](https://static.igem.org/mediawiki/2020/8/83/T--UofUppsala--TypeIIS_boxplot.png)

**Figure 9.** Box plot of the standardized amount of DNA (y) depending on the copy number (x)

## Creation of Dummy Parts

![Detail of pSB3C11-DY sequencing.](https://static.igem.org/mediawiki/2020/d/d9/T--UofUppsala--pSB3C11-DY.png)

**Figure 10.** Detail of pSB3C11-DY sequencing. Annotated is the dummy part itself as well as fusion sites and BsaI recognition sequences.

Type IIS iGEM standard allows for fast and efficient construction of transcriptional units (TU) and multi-transcriptional units (MTU) in groups of four components. These components are needed in Level 1 assemblies (promoter, RBS, CDS and terminator) but might not be needed in higher level assemblies if the amount of transcriptional units in your system is not a multiple of four.

For this reason, placeholders called dummy parts were created based on an existing Modular Assembly method [(4)](#(4)). These parts mimic a basic Type IIS part or a transcriptional unit and can be included in the assembly whenever needed. Two different dummies (TU-DY (BBa\_K3425017) and MTU-DY (BBa\_K3425018)) were ordered as primers with the corresponding fusion sites to be cloned into all different pOdd and pEven plasmids, with four nucleotides (AACA) in between.

Therefore, pSB1K01-DY is the dummy cloned into pSB1K01 which mimics TU1. It can be cloned to a pEven plasmid like pSB3C11 together with other transcription units or dummies which represent TU2, TU3 and TU4. pSB3C11-DY mimics MTU1 or a promoter part and it can be cloned to a pOdd plasmid in a similar way.

![Detail of the sequencing result of the  multi-dummy cloning  (TU-DY^4) into pSB3C11.](https://static.igem.org/mediawiki/2020/0/0f/T--UofUppsala--DY4_pSB3C11.png)

**Figure 11.** Detail of the sequencing result of the multi-dummy cloning (TU-DY^4) into pSB3C11. At the upper part, dummy parts, fusion sites and BsaI recognition sequences are annotated. At the bottom, three sequencing reads corresponding to three different clones are shown.

In **Figure 10**, the sequencing of pSB3C11-DY can be seen as one example of these dummy parts. All the dummy clonings were successful, and the results can be found in the corresponding [Parts](https://2020.igem.org/Team%3AUofUppsala/Parts).

In order to check whether these would work as dummy parts, all four pSB1K0#-DY mimicking four TUs were cloned together into pSB3C11. Similarly all four dummies mimicking MTUs pSB3C1#-DY were cloned into pSB1K02. Sequencing (**Figures 11** and **12**) revealed that only one out of six clones contains the expected sequence (the last clone of **Figure 11**). The remaining five clones contain deletions or insertions of various sizes, all of them in the area of the “multi-dummy” sequence.

This is probably due to the short and repetitive sequence generated by the dummies, which might have caused polymerase slippage. This phenomenon can occur when there are reiterated sequences, even if they are imperfect [(5)](#(5)). Since the fusion sites at the ends, next to the BsaI and SapI recognition sequences, are present in all the clones it is likely that the cloning was successful and the polymerase slippage happened afterwards, during the growth of the transformed cells.

![Detail of the sequencing result of the multi-dummy cloning  (MTU-DY^4) into pSB1K02.](https://static.igem.org/mediawiki/2020/3/38/T--UofUppsala--DY4_pSB1K02.png)

**Figure 12.** Detail of the sequencing result of the multi-dummy cloning (MTU-DY^4) into pSB1K02. At the upper part, dummy parts, fusion sites and BsaI recognition sequences are annotated. At the bottom, three sequencing reads corresponding to three different clones are shown.

Dummy parts are only supposed to be fillers that allow the cloning reaction to ligate properly when you have less than four parts to assemble. In this sense, this dummy sequence works as intended. However, it is not ideal to have unpredictable mutations occurring systematically.

A simple solution would be not cloning the dummy sequences next to each other. There might be a situation where this is not possible, so in that case it might be good to use longer dummy parts that are all different from each other. These parts were designed *in silico* by retrieving random 8bp DNA sequences from the Random DNA Generator [(6)](#(6)) and adding the corresponding fusion sites. They were added in the parts registry as pSB1K0#-DY (BBa\_K3425023 to 26) and pSB3C11 (BBa\_K3425027 to 30).

[Development of Type IIS Notebook](https://static.igem.org/mediawiki/2020/e/ec/T--UofUppsala--TypeIIS_NB.pdf)

# First Generation

*Development of DNA binding domain-based constructs for an operative nanobody-based caffeine-detection system.*

![](https://static.igem.org/mediawiki/2020/9/97/T--UofUppsala--arrow-lab.svg)

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## DNA Binding Domains

In our minimal system, we use DNA binding domains (DBDs) for analyte detection as they are naturally occurring in *Escherichia coli* ( *E. coli*). The DBDs presented here are designed to get activated and induce transcription of downstream genes upon the presence of caffeine. To test the design, we performed the following experiments.

#### Assemblies of pSB1C3+DBD-Caff, pSB3K3+pCad and Double Transformations

For cloning of the two constructs pSB1C3+DBD-Caff, pSB3K3+pCadBA+mRFP we used the Gibson assembly method.

DBD-Caff was assembled with pSB1C3 (see Notebook), a high copy number plasmid. The construct was transformed into the  *E. coli*  strain DH5α as well as into the  *E. coli*  Top10 strain. The presence of the plasmid was confirmed by colony PCR for two colonies of each strain. However, the sequencing results after miniprep showed a frameshift mutation for the plasmid of DH5α cells, and the experiment was henceforth continued with the Top10 strain.

The operator pCadBA was assembled with mRFP and pSB3K3 (see Notebook), a low copy number plasmid. The expression of mRFP is therefore controlled by the binding of DBD-Caff to the pCadBA operator upon the presence of caffeine. After transformation of the plasmid into DH5α, ten colonies were inspected. Several of these colonies were sequenced and confirmed as having assembled the desired construct.

The two assembled constructs pSB1C3+DBD-Caff and pSB3K3+pCadBA were then used for double transformation (see Notebook). The gel electrophoresis of colony PCR products and sequencing results showed that the double transformation was successful. However, the colonies of the double transformation appeared still red. This indicates the constitutive expression of mRFP without induction. The expected results were white colonies with very low to no expression of mRFP in absence of caffeine and red colonies with high mRFP expression in the presence of caffeine. Comparing our system to the system presented in the paper, we noticed that the expression of DBD-Caff in our construct is much higher [(7)](#(7)). The high expression level of DBD-Caff derives from a strong promoter in a high copy number plasmid. This design can reasonably cause non-specific activation of the pCadBA promoter. Taking this explanation as the working hypothesis, the next objective was to reduce the expression of DBD-Caff to reduce the non-specific activation of the expression of mRFP, and increase the copy number of pCadBA to increase the range of expression of mRFP.

#### Subcloning of pSB3K3+DBD-CaFF and pSB1C3+pCadBA

Due to the result of the double transformation, the previously assembled plasmids underwent subcloning (see Notebook). The results were confirmed by a change in the phenotype (**Figure 13B**). The colonies containing both plasmids had soft-red colour, and adding caffeine increased the expression of mRFP in a visually appreciable manner. The increased expression of mRFP was observed in several colonies.

![](https://static.igem.org/mediawiki/2020/f/f1/T--UofUppsala--fig13_wetlab.jpg)

**Figure 13.** Results of the subcloning. Picture A shows the 1% Agarose Gel Electrophoresis (stained with SYBR Safe) of colony PCR products of the eight colonies (1-8). Wells 1-4 are the subcloned pSB3K3+DBD-CaFF and well 5-8 pSB1C3+pCadBA. Using standard primers VF2 and VR, the expected amplification product for both plasmids was around 1200bp, which was the case. Picture B shows the change in phenotype where the Falcon tube on the left is a DH5α culture with pSB3K3+DBD-Caff and pSB1C3+pCadBA with 1000μM caffeine and the tube on the right is the same culture but without caffeine.

#### Caffeine Experiments

A caffeine assay was also performed (see Notebook). The molecule detection system that has been designed is a basic example in which caffeine is used as the analyte of interest. The presence of caffeine in the environment triggers the activation of the system.

The proof that the system is inducible under presence of caffeine is shown in [Proof of concept](https://2020.igem.org/Team%3AUofUppsala/Proof_Of_Concept).

[DBDs Notebook](https://static.igem.org/mediawiki/2020/5/5b/T--UofUppsala--DBDs_NB.pdf)

# Next Generation

*Efforts on establishing the two-component system NarX-NarL in Escherichia coli and Bacillus subtilis, assembling of signal amplification and noise reduction components, and attempts on developing a biosensor based on agglutination*

![](https://static.igem.org/mediawiki/2020/9/97/T--UofUppsala--arrow-lab.svg)

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### Detection Module with an Integrated Amplification Step

#### Testing the System in *Escherichia coli*

As a proof-of-concept of a functioning detection system, the aim was to design and express an anti-caffeine nanobody (VHHac) fused to the transmembrane domain of the histidine kinase NarX in  *E. coli*. When two VHHac bind to caffeine, it brings two VHHac-NarX molecules in close proximity, activating NarX through phosphorylation. This in turn phosphorylates the response regulator, NarL, which binds to and activates its respective promoter PyeaR. In our designed system, PyeaR acts as a promoter for the GFP coding sequence thus, GFP will be expressed when caffeine is present. After developing the proof-of-concept in *E. coli*, the system would be tested and implemented in *B. subtilis*.

In order to execute this, we designed a sequence consisting of PyeaR with GFP and two copies VHHac fused to NarX, each with the constitutive promoter BBa\_J23100. The ribosomal binding site for all three proteins was BBa\_B0034. The transcriptional terminator of GFP was BB1\_B0010. The design of the system also consists of NarL, which is constitutively expressed and intrinsically present in *E. coli*. In order to make the construct compatible with Gibson Assembly, it was designed with complementary sequences to the pSB1C3 backbone plasmid. Thus, three Gibson Assembly fragments consisting of PyeaR-GFP and two copies of VHHac-NarX was designed (**Figure 14**).

![](https://static.igem.org/mediawiki/2020/3/35/T--UofUppsala--NarX_construct.png)

**Figure 14.** PyeaR\_GFP + 2x VHH-NarX construct. A GFP is under the control of the yeaR promoter, while the 2 copies of VHH-NarX are constitutively expressed. This construct is flanked by the Biobrick prefix and suffix, as well as by pSB1C3 homology regions (adaptors necessary for the Gibson Assembly). The 3 RBSs (B0034), terminators (BBa\_B0010) and Biobrick scars are not shown for visual clarity.

A second goal was to optimize the system in *E. coli* by introducing two different point mutations in NarX to reduce noise. Since the NarX kinases dimerize upon proximity, Mazé and Benenson [(8)](#(8)) introduced a system where two different point mutations are introduced in separate NarX kinase proteins in order to reduce phosphorylation between kinases. Thus, the two copies of VHHac-NarX were differentiated by codon optimization in order to perform site-directed mutagenesis.

![](https://static.igem.org/mediawiki/2020/f/f9/T--UofUppsala--BB_Colony_PCR.jpg)

**Figure 15.** 1% Agarose Gel Electrophoresis (stained with SYBR Safe) of colony PCR product. Wells “O” and “Q” are colony PCR products from transformed cells. Wells marked as \* contain colony PCR products from non-transformed colonies. The ladder used was GeneRuler 1kb Plus DNA Ladder.

In order to express and test the system in *E. coli* the first step was to purify the pSB1C3 plasmid followed by PCR linearization (see Purifying pSB1C3 in Notebook). To assemble the three fragments with the backbone of pSB1C3, Gibson Assembly was performed (see Gibson Assembly in Notebook) and the product was transformed into *E. coli* (see Transformation in Notebook).

To verify that the system was transformed, colony PCR was performed on a number of colonies (see colony PCR in Notebook) followed by gel electrophoresis (see Gel Electrophoresis in Notebook). The gel electrophoresis indicated that the product of two colonies, referred to as “O” and ”Q” was closer to the desired 3.3 kb than other products (**Figure 15**). Although the products were less than 3.3 kb, they were observed under UV light where colony “O” showed fluorescence indicating a successful transformation of GFP (**Figure 16**). However, GFP was expressed without the presence of caffeine, indicating leakage.

![](https://static.igem.org/mediawiki/2020/1/1c/T--UofUppsala--Ecoli_NarXNarL_GFP.jpg)

**Figure 16.** Transformed colonies under UV light.

To further investigate these colonies, the transformed products were sequenced (see Sequencing in Notebook). The results showed two alignments of primers VF2 and VR, meaning that two of the three fragments were successfully integrated into pSB1C3 and were transformed into the cell.

Additionally, during the Gibson Assembly, the first copy of VHH-NarX was almost entirely deleted. Thus, the resulting construct comprised the PyeaR-GFP and only the second copy of VHH-NarX (**Figure 17**). After further analyzing the sequence, we found out that the second fragment of the Gibson Assembly reaction had homology to itself. Therefore, since this assembly method is dependent on sequence homology for joining the fragments together, these regions may have annealed with each other during the reaction, causing a "self-excision" by homologous recombination. Luckily, this did not affect the sequence of VHH-NarX: it solely implies that there is one copy of this CDS instead of the intended two copies. This also explains why the gel showed a product of 2.4 kb, corresponding to only one copy construct.

Three caffeine assays were performed on colonies “O” and “Q” (see Caffeine Assay in Notebook) in order to test the system. In the first assay, all cultures including the one without caffeine showed fluorescence and the intensity and absorbance were independent of the caffeine concentration. The hypothesis was that activation of the natural NarX/NarL two-component system had occured due to the presence of nitrates and nitrites in the LB-medium. Another caffeine assay using M9-medium was performed though with no conclusive results, partially explained by the slow cell growth and inability to reach OD 0.3. The use of a high copy number plasmid might be the explanation of the constant expression of GFP, leading to leakage. Thus, future experiments to improve the system may be to change to a low copy number plasmid and only design one copy of the desired genes in the construct to avoid self-excision.

![](https://static.igem.org/mediawiki/2020/d/df/T--UofUppsala--NarXVHH_sequencing.png)

**Figure 17.** PyeaR\_GFP+2xVHH-NarX sequencing results (Colony “O”). The figure depicts a sequence alignment where the sequencing results of colony “O” (represented by the red rectangles) are aligned to the sequence of the construct we were aiming for. It is possible to conclude that the plasmid present in this colony is missing the first copy of VHH-NarX.

[NarX/NarL in *E. coli* Notebook](https://static.igem.org/mediawiki/2020/a/a4/T--UofUppsala--NarXNarL_Ecoli_NB.pdf)

## Gram-positive Host

#### Transformation of the Module in *Bacillus subtilis*

Before transformation, the  *B. subtilis*  plasmids pBS1C and pBS2E were submitted to PCR amplification, to linearize them and remove the mRFP that these plasmids originally contained. Afterwards, the Detection Module constructs were inserted in the respective vectors through Gibson Assembly.

A *B. subtilis* culture was then transformed\* with both plasmids at the same time and plated on LB-agar supplemented with the appropriate concentrations of Erythromycin, Lincomycin and Chloramphenicol. On the following day, no growth was observed in these plates. So, a two-step approach was adopted: *B. subtilis* was first transformed with pBS1C; upon overnight incubation in Chloramphenicol plates, 6 colonies were picked, submitted to colony PCR (to confirm the presence of PydfJ115\_GFP) and then transformed with pBS2E and respective insert.

After plating in double resistance LB-plates, each plate yielded 1-3 colonies, including a negative control where a strain transformed only with pBS1C was inoculated in double resistance plates, pointing towards a failed transformation. This was further confirmed by a colony PCR, where the results obtained were inconclusive.

We suspect that the negative outcome of pBS2E transformation may have been caused by the toxicity of this plasmid’s insert (Rewired NarL + VHH-NarX). A possible explanation for growth inhibition of transformed cells could stem from the size of the VHH-NarX protein alongside the strength of the respective constitutive promoter.

\*The *B. subtilis* transformation protocol used by our team – ISN 2.0 – is a more consistent and practical version of another protocol. If you would like to have access to the original protocol and know more about the optimization process that led to this current version, please check the [*B. subtilis* Handbook](https://static.igem.org/mediawiki/2020/5/50/T--Brno_Czech_Republic--Contribution_Handbook.pdf), done as a collaboration with iGEM team Generation Mendel, from Brno University, Czech Republic.

[NarX/NarL in *B. subtilis* Notebook](https://static.igem.org/mediawiki/2020/8/80/T--UofUppsala--NarXNarL_Bsubtilis_NB.pdf)

## Signal Amplification and Noise Reduction

As described above, the presence of target ligand specifically activates the detection module and triggers the expression of the reporter protein, β-galactosidase (β-gal). The related experiments showed that essential components of the "minimal biosensor" work as expected. However, they also confirmed our initial hypothesis that at the absence of the ligand there is a background signal - noise (see [Proof of concept](https://2020.igem.org/Team%3AUofUppsala/Proof_Of_Concept)). To increase the signal to noise ratio, the system needs to be optimised. This can be achieved by implementing several signal amplification and noise cancelling mechanisms. As we have foreseen this issue, we have already started building components for optimization.

### Reporter System: **β**-galactosidase

Our reporter system is based on the enzymatic cleavage of X-gal or ONPG by β-galactosidase.

![](https://static.igem.org/mediawiki/2020/b/b6/T--UofUppsala--OD_XGal_ONPG.jpg)

**Figure 18.** Colour change of media by X-gal ONPG cleaved by β-galactosidase  *in vitro* . Substrate with increasing concentration of β-galactosidase in 0.5 U/ml steps from left to right. (A) X-gal as a substrate. (B) ONPG as a substrate.

Before testing the β-galactosidase activity  *in vivo* we tested the reaction time of the enzyme  *in vitro*. For this, different concentrations of β-galactosidase ranging from 0.5 to 3 U/ml were added to 0.2 mg/ml X-gal or ONPG. The absorbance was measured for 1 h at OD610 for X-gal or at OD420 for ONPG. From the values of three biological replicates a standard curve was created. By mixing β-galactosidase with X-gal  *in vitro* , a visible blue color change of the media could be observed in approximately 30-40 minutes (**Figure 19**), while cleavage of ONPG resulted in a yellow colored media in 5-10 minutes (**Figure 20**). The absorbance for the  *in vitro*  samples in ONPG or X-gal raised with increasing amounts of β-galactosidase as expected. This is also visible by naked eye as shown in **Figure 18.** Despite the faster colour change using ONPG as a substrate we decided to use X-gal for the final system because yellow is harder to see by naked eye as the color of the culture media is slightly yellow.

![](https://static.igem.org/mediawiki/2020/2/24/T--UofUppsala--invitroXGal.png)

**Figure 19.** Absorbance of X-gal cleaved by β-galactosidase  *in vitro* . Graph showing absorbance comparison of a range of enzyme concentrations (0.5 - 3 U/ml) in 0.2 mg/ml X-gal. Absorbance was read at 610 nm. The color change to translucid-blue was visible between 30-40 min depending on the concentration.

![](https://static.igem.org/mediawiki/2020/6/6f/T--UofUppsala--invitroONPG.png)

**Figure 20.** Absorbance of ONPG cleaved by β-galactosidase  *in vitro* . Graph showing absorbance comparison of a range of enzyme concentrations (0.5 - 3 U/ml) in 0.2 mg/ml ONPG. Absorbance was read at 420 nm. The color change to translucid-yellow was visible between 5-10 min depending on the concentration.

For the expression of β-galactosidase *in vivo* its corresponding gene lacZ was amplified from the *E. coli* MG1655 chromosome via PCR. The agarose gel electrophoresis of the PCR product is shown in **Figure 21**. The band indicates a DNA fragment length of > 3000bp which resembles the size of lacZ (3075bp).

![](https://static.igem.org/mediawiki/2020/7/74/T--UofUppsala--extraction_gel.jpg)

**Figure 21.** 1% Agarose Gel Electrophoresis (stained with SYBR Safe) of LacZ amplified from the genome of  *E. coli*  MG1655.

![](https://static.igem.org/mediawiki/2020/7/7d/T--UofUppsala--GB_TIIS_gel.jpg)

**Figure 22.** 1% Agarose Gel Electrophoresis (stained with SYBR Safe) of the amplified target gene after Gibson and Type IIS assembly. Loaded from left to right: 1 kb Plus DNA Ladder, pBAD24 plus LacZ Gibson assembly clone 1, pBAD24 plus LacZ Gibson assembly clone 2, pSB1K01 plus LacZ Type IIS clone 1, pSB1K01 plus LacZ Type IIS clone 2.

For cloning, two different methods were used: Type IIS assembly and Gibson assembly. With Type IIS assembly lacZ was cloned into the pSB1K01 plasmid combined with a constitutive promoter. With Gibson assembly lacZ was cloned into pBAD24 which includes the inducible pBAD promoter. Both plasmids were transformed into chemically competent *E. coli* DH5α cells. We chose this strain as our model organism as it lacks the natural chromosomal expression of β-galactosidase to avoid false positive reactions.

LacZ was only inserted correctly into pBAD24. Type IIS assembly was implemented several times but remained unsuccessful. **Figure 22** shows the gel electrophoresis of the colony PCR products from the target genes after the plasmids were cloned and transformed. The second of the two clones transformed with pBAD24 shows a band at the size of 3000 bp which indicates the insertion of lacZ.

The DNA sizes in lane 3 and 4 indicate an unspecific insertion of DNA fragments and ligation of pSB1K01. Vaguely speculating, the reason for the difficult integration of lacZ into pSB1K01 via Type IIS could reside in the length of the gene.

The successfully transformed cells were subjected to 1 mg/ml X-gal or ONPG. First, cells grew in LB media mixed with glucose overnight, and were diluted the next morning. Glucose silences the expression of lacZ. To start the expression of lacZ 0.2% arabinose was added. As a negative control CaFF-I pSB1C3 DH5α cells were used. To test if X-gal/ ONPG was cleaved intra- or extracellular, the reactant was added to the cells directly or to the supernatant after centrifugation of the cells. Absorbance was measured with a spectrophotometer every 30 minutes for a total of 180 minute. **Figure 23** shows that the change in absorbance is higher for ONPG than for X-gal. A visible color change was observed only for ONPG samples after 60 minutes.

The absorbance measurements of the  *in vitro*  and  *in vivo*  experiments could not be compared. The reason for this is that for the  *in vivo*  samples no colour change could be observed by naked eye when adding 0.2 mg/ml substrate as we did in the  *in vitro*  measurements. Therefore, 1 mg/ml of X-gal/ ONPG was added to the cells.

![](https://static.igem.org/mediawiki/2020/8/8c/T--UofUppsala--invivopBAD24.png)

**Figure 23.** Absorbance of LacZ expressed  *in vivo*  in media mixed with ONPG or X-gal. Expression of LacZ was induced by arabinose. Cells grew in media and were normalized to growth rate. Negative control samples lack the expression of β-gal.

![](https://static.igem.org/mediawiki/2020/b/b3/T--UofUppsala--bgal_secretion_test.jpg)

**Figure 24.** Expression of β-galactosidase in X-gal or ONPG. From left to right. cells in LB, cells in LB plus X-gal, cells in LB plus ONPG. (A) Negative control, non-β-galactosidase expressing DH5α. (B) pBAD24-lacZ DH5α.

In order to achieve an even stronger signal, the final step would have been to work on the compatibility of the β-galactosidase system with the rest of the amplification loop. The β-galactosidase system was not combined with the detection system due to lack of time. The reporters used for testing the system were RFP and GFP.

[β-galactosidase Notebook](https://static.igem.org/mediawiki/2020/b/b3/T--UofUppsala--BetaGal_NB.pdf)

### Q**β** Replicase Amplification System

#### Q**β**

![Cloning of Qβ replicase β-subunit](https://static.igem.org/mediawiki/2020/8/87/T--UofUppsala--QB_replicase.jpg)

**Figure 25.** Cloning of Qβ replicase β-subunit. When expressed constitutively, β-subunit of the Qβ replicase is probably toxic and therefore mutates, as seen from the two independent cases of genomic DNA insertion (A). In line with this, cloning of β-subunit under inducible promoter pCAD did not result in any mutations (B).

To increase the sensitivity of the system, Qβ replicase was introduced (see Design). Initially, the β-subunit of Qβ replicase was cloned under a constitutive promoter to simply test the protein expression. Since this resulted in large insertions of genomic DNA into the Qβ sequence (**Figure 25A**), Qβ is likely toxic to the cell in the amount we tried to express it in. Previously, Yao  *et al.*  [(9)](#(9)) expressed Qβ under an inducible promoter. Therefore in the next step, we cloned Qβ replicase under the pCAD inducible promoter (**Figure 25B**), which prevented Qβ from mutating. pCAD is the promoter activated by the detection module and linking it directly to QB replicase expression is the simplest variant of incorporating the Qβ amplification system in the biosensor. This construct is needed for testing the Qβ amplification. However, in the final design, Qβ is controlled by T7 RNA polymerase, which allows for implementing the T7 lysozyme as the noise reducing mechanism.

#### Q**β** Recognition Sequences

Qβ requires specific recognition sequences on either side of the sequence you want to amplify. 5'Qβ and 3'Qβ were shown to be the most efficient pair of recognition sequences [(9)](#(9)) and therefore we chose to use these.

Type IIS iGEM assembly standard allows easy cloning of transcriptional units which consist of four typical parts - promoter, RBS, CDS and terminator (You can read more about Type IIS [here](https://2020.igem.org/Team%3AUofUppsala/Engineering#TypeIIS).) However, the template of Qβ replicase is atypical in a sense that it is composed of more than four parts: promoter, 5'Qβ recognition site, RBS, CDS, 3'Qβ recognition site and terminator. To simplify the cloning procedure for initial experiments, the 5' recognition site was ordered together with medium strength RBS (B0034) and 3' recognition sequence was fused with the terminator obtained from the Distribution Kit 2019.

![3'Qβ recognition site](https://static.igem.org/mediawiki/2020/b/ba/T--UofUppsala--QB_3.jpg)

**Figure 26.** 3'Qβ recognition site is prone to mutate in the specific region around 115 nt from its 5' end. Deletions in fusion construct of 3' recognition sequence with the terminator BBa\_B0015 (A) as well as in 3' recognition sequence cloned to pSB1C00 Level 0 backbone (B) are shown.

Since synthesis of terminator sequences is non-trivial, the 3´ recognition site was synthesised separately and then fused to the terminator obtained from the Distribution Kit 2019 (see Notebook) .

When the fusion construct of 3´site and terminator was sequenced, deletions of 29bp were found in the middle of 3' recognition site (**Figure 26A**). Since the 3´ recognition site was synthesised, we first suspected that the mutations emerged during PCR (Notebook). However, subsequent sequencing of the 3' recognition site cloned alone into Level 0 backbone revealed the set of non-identical deletions of 24 or 31 bp localised exactly in the same region (**Figure 26B**). Since the 3' recognition site is an RNA functional element, any change in its sequence might compromise its function. The sequence itself seems to be prone to mutate. Since in Level 0 the 3' recognition sequence is not transcribed, the mutation pressure probably acts on the DNA level. Without a working set of two recognition sequences, no further experiments measuring the activity of Qβ could be done.

### Noise Reduction

#### TEV Protease

In order to reduce the basal level of the Qβ replicase, we designed the Qβ sequence with the LAA degradation tag at the 3’ end. In between the Qβ sequence and the degradation tag, a TEV cleavage site was inserted. This would allow to cut off the degradation tag by TEV protease when the system is activated resulting in a stable level of Qβ replicase.

The TEV protease sequence was ordered with the Type IIS CDS prefix and suffix and it was cloned into the Level 0 backbone. However, we did not proceed further with the implementation of the TEV protease into our system.

#### T7 RNA Polymerase and T7 Lysozyme

![T7 Lysozyme sequencing detail](https://static.igem.org/mediawiki/2020/5/53/T--UofUppsala--lysozyme.png)

**Figure 27.** Glycine to valine substitution at position 119 of the T7 lysozyme in Level 1 constructs.

Apart from adding TEV tag to avoid leaking problem, more approaches were added. Since the Qβ replicase is highly sensitive, even a small amount of leakiness will be amplified and can cause a false-positive result. To obstruct this condition, T7 RNA polymerase was introduced to express Qβ. T7 RNA polymerase is extremely promoter-specific and more importantly, it can be reliably controlled by T7 lysozyme. This makes the T7 lysozyme a key component of the noise-reducing mechanisms.

In the final design, we place a constitutive promoter before the T7 lysozyme, so it is produced at a stable level and blocks the activity of the basal level of T7 RNA polymerase. The basal level of T7 RNA polymerase was determined by the level of noise measured in the detection module (see [Proof of concept](https://2020.igem.org/Team%3AUofUppsala/Proof_Of_Concept)). Subsequently, the level of T7 lysozyme would be set so as to be higher than that, in order to block all T7 RNA polymerase molecules.

T7 RNA polymerase and T7 lysozyme were amplified from the *E. coli*(BL21)pLysS (Notebook) and the size was verified by agarose gel electrophoresis. For the purpose of experiments verifying the expression of T7 lysozyme, we first cloned it under LacUV5 and T7M5 inducible promoters. Since single amino acid of change was identified in both clones [Gly(119)->Val(119)] work was not continued due to the time constraints.

[Qβ and Noise Reduction Notebook](https://static.igem.org/mediawiki/2020/9/92/T--UofUppsala--QB_Noise_NB.pdf)

## Checking Nanobodies: Agglutination Approach

With the agglutination approach we wanted to develop a biosensor based on an agglutination reaction using caffeine-binding nanobodies. These nanobodies are recombinant antibodies (VHH) and bind specific to caffeine. Agglutination can be triggered in the presence of large antigens such as BSA-caffeine haptens. Caffeine can compete for the binding of nanobodies, stopping agglutination from happening and triggers pelleting of the cells instead. The agglutination test as described by Riangrungroj et al. [(10)](#(10)) aims to figure out the amount of caffeine that occupies enough nanobodies to trigger clumping. This reaction serves the function of a biosensor.

For detecting the minimal concentration, anti-caffeine VHH expressing cells where opposed to BSA-caffeine as well as to different concentrations of caffeine. When BSA-caffeine binds to the nanobodies, cells will agglutinate as BSA serves as a space holder between the  *E. coli*  cells. When a higher concentration of caffeine than BSA-caffeine is present in the sample, caffeine will interact with the nanobodies instead. Caffeine as a much smaller molecule reduces the space between the cells consequently resulting in a cell pellet.

![Agglutination approach](https://static.igem.org/mediawiki/2020/1/12/T--UofUppsala--agglutination_new.svg)

**Figure 28.** Single E. coli cells express the VHH specific for caffeine binding. By adding caffeine to the cells, a cell pellet forms. By adding BSA-caffeine, cells agglutinate to each other. By adding a higher concentration of caffeine than BSA-caffeine to the cells, caffeine is more likely to be bound leading to a cell pellet.

For performing the experiments, the nanobodies were designed to be placed in the outer membrane of *E. coli*  DH5α cells. Therefore, anti-caffeine VHH was attached to intimin which is a cell membrane protein and important for the transportation of the nanobody to the outer membrane. To express this fusion protein called CaFF-I, the coding sequence was cloned into the high copy number plasmid pSB1C3. Subsequent, the construct was transformed into chemically competent *E. coli* DH5α cells for amplification and storage.
However, the sequence of CaFF-I showed frame-shifting mutations repeatedly after sequencing. The difficulty of the assembly and the mutations indicate that this construct might be toxic to the cell when overexpressed constitutively.

The next steps to attain a successful agglutination biosensor are:

* Use inverse mutagenic PCR to fix the frameshift mutations
* Redo assembly, accounting for transformation efficiency of a control. This will give us more knowledge on the possible toxicity of CaFF-I
* Reassemble CaFF-I on an inducible vector (e.g. pET)
* Develop an assay to measure the binding affinity of the nanobody to caffeine
* Once the previous steps are successful, determine the Limit Of Detection (LOD), the lowest concentration that our cellular biosensor is capable of detecting

Detailed protocols for experimental implementation can be accessed below.

# Protocols

![](https://static.igem.org/mediawiki/2020/9/97/T--UofUppsala--arrow-lab.svg)

---

| Download | Protocol |
| --- | --- |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/9/96/T--UofUppsala--Agarose_new2.pdf) | Agarose Gel Electrophoresis |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/5/50/T--Brno_Czech_Republic--Contribution_Handbook.pdf) | ISN 2.0 Transformation Protocol (*B. subtilis* Handbook, pages 16 and 17) |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/9/93/T--UofUppsala--Caffeine_assay_protocol.pdf) | Caffeine assay |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/0/07/T--UofUppsala--Cryo-stock.pdf) | Cryostock |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/7/73/T--UofUppsala--Determination_new2.pdf) | Determination in vitro of B-gal activity |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/b/be/T--UofUppsala--Development_of_Competence_in_Bacillus_subtilis_and_Transformation.pdf) | Development of Competence in *B. subtilis* and Transformation |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/5/5b/T--UofUppsala--Distribution_kit_DNA_resuspension.pdf) | Distribution kit DNA resuspension |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/8/8b/T--UofUppsala--Electroporation_-_Transformation_of_B._subtilis_.pdf) | Electroporation - Transformation of *B. subtilis* |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/d/d5/T--UofUppsala--Eurofins_Mix2seq_kit_.pdf) | Eurofins Mix2seq kit |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/1/14/T--UofUppsala--GeneJET_PCR_Purification_Kit%2C_K0701.pdf) | GeneJET PCR Purification Kit, K0701 |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/9/93/T--UofUppsala--GeneJET_Plasmid_Miniprep_Kit%2C_K0502.pdf) | GeneJET Plasmid Miniprep Kit, K0502 |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/d/de/T--UofUppsala--Genomic_DNA_Purification.pdf) | Genomic DNA purification |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/2/25/T--UofUppsala--Gibson_Assembly.pdf) | Gibson Assembly |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/7/7e/T--UofUppsala--Lyophilization.pdf) | Lyophilization |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/4/4d/T--UofUppsala--LB_media_new2.pdf) | Luria-Bertani (LB) media liquid and agar |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/d/d7/T--UofUppsala--NEBuilder_new2.pdf) | NEBuilder HiFi DNA Assembly Reaction |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/6/66/T--UofUppsala--Overnight_culture.pdf) | Overnight liquid culture |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/5/56/T--UofUppsala--PCR_-_Phusion%E2%84%A2_High%E2%80%93Fidelity_DNA_Polymerase.pdf) | PCR - Phusion™ High–Fidelity DNA Polymerase |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/a/a9/T--UofUppsala--PCR_reaction_new2.pdf) | PCR reaction - Phusion and Colony PCR |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/6/62/T--UofUppsala--Plasmid_Isolation.pdf) | Plasmid Isolation |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/4/40/T--UofUppsala--Promegakit_new2.pdf) | Promegakit - Assembly of Restriction Enzymes Digestion |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/1/1f/T--UofUppsala--Sample_Preparation_for_Bacillus_subtilis_Colony_PCR.pdf) | Sample Preparation for *B. subtilis* - Colony PCR |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/2/24/T--UofUppsala--Sequential_Double_Digest.pdf) | Sequential Double Digest |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/d/de/T--UofUppsala--Spheroplast_new2.pdf) | Spheroplasts formation |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/6/68/T--UofUppsala--Subcloning_new2.pdf) | Subcloning: digestion and ligation reaction |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/5/58/T--UofUppsala--Transformation_new2.pdf) | Transformation of chemically competent *E. coli* DH5α cells |
| [![](https://static.igem.org/mediawiki/2020/a/ac/T--UofUppsala--download.png)](https://static.igem.org/mediawiki/2020/e/e6/T--UofUppsala--Type_IIS_cloning.pdf) | Type IIS cloning (MoClo) |

# References

---

1. Jones, K. L., Kim, S.-W., and Keasling, J. D. (2000) Low-Copy Plasmids can Perform as Well as or Better Than High-Copy Plasmids for Metabolic Engineering of Bacteria. Metabolic Engineering. 2, 328–338
2. How do I know if my plasmid is a high- or low copy number type? - QIAGEN [online] https://www.qiagen.com/no/resources/faq?id=1f42840e-fbd7-4734-b0cd-e17372a9e5a4&lang=en (Accessed October 24, 2020)
3. CopyRight v2.0 BAC Cloning Kits (pSMART BAC and pEZ BAC) [online] https://www.lucigen.com/CopyRight-v2.0-BAC-Cloning-Kits-pSMART-BAC-and-pEZ-BAC/#subcat-tabs3 (Accessed October 21, 2020)
4. Binder, A., Lambert, J., Morbitzer, R., Popp, C., Ott, T., Lahaye, T., and Parniske, M. (2014) A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants. PLOS ONE. 9, e88218
5. Levinson, G., and Gutman, G. A. (1987) Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol Biol Evol. 4, 203–221
6. Random DNA Generator [online] https://www.faculty.ucr.edu/~mmaduro/random.htm (Accessed October 16, 2020)
7. Chang, H. J., Mayonove, P., Zavala, A., De Visch, A., Minard, P., Cohen-Gonsaud, M., and Bonnet, J. (2018). A modular receptor platform to expand the sensing repertoire of bacteria. ACS synthetic biology, 7(1), 166-175.
8. Mazé, A., and Benenson, Y. (2019) Artificial signaling in mammalian cells enabled by prokaryotic two-component system. Nat Chem Biol. 16, 179–187
9. Yao, Y., Zhang, W., Zhang, M., Jin, S., Guo, Y., Zu, Y., Ren, K., Wang, K., Chen, G., Lou, C., and Wu, Q. (2019) A Direct RNA-to-RNA Replication System for Enhanced Gene Expression in Bacteria. ACS Synth. Biol. 8, 1067–1078
10. Riangrungroj, P., Bever, C. S., Hammock, B. D., and Polizzi, K. M. (2019). A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid insecticide exposure. Scientific reports. 9(1), 1-9

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