In 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) and Loop assembly (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. Other pages such as the Type IIS backbone registry pages (for example pSB1C00 ) and the MoClo (1) and Loop (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.

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 and in the registry.

Dummy parts

When we started working with the iGEM Type IIS standard, we realised the need to have dummy parts. Thus, we decided to create these parts since the beginning, and 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).

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).

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) 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.

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). 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).

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.

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.

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 NANOFLEX kit

1. Bacteria chamber: the chamber would hold the lyophilized bacteria that we have engineered, separate from the media. In the case of the reporter being an enzyme, the chamber would hold the substrate as well, i.e. in case of beta-galactosidase being the reporter enzyme this chamber would hold X-gal. Upon the use of the kit the media would have to enter this chamber and induce growth. As the test results are to be seen by the naked eye, this chamber has to be transparent.

2. Media chamber: for holding sterile media.

3. Sample chamber: this is the chamber in contact with the user. Upon use of the kit the user would place the sample (blood sample/sputum smear diluted in water) in this chamber. The design needs to ensure the sample coming in contact with the bacteria chamber.

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.

Users' 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 achieve effective sealing in many chemical/biological products. However, if the membrane is exposed to the user, they can easily be manipulated and kit contents may be used as not intended. 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.

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.

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 locking mechanism with 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.

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.

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.