Team:UofUppsala/Engineering

Engineering Success

Index

• Development of iGEM Type IIS standard
• Prototype of NANOFLEX hardware
• 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

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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!

iGEM Type IIS guide

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 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).

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

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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.

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

Key Concepts Dictating Kit Design

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.

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.

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.

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

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

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.

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.

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.

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.

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

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