Every iGEM project contributes towards interest and advancement in synthetic biology. However the transition from an interesting lab project to a commercial product is a big step. We believe that DNA will become a long-term storage medium, and so it is important to look to the future of this being implemented, and show how our system can be used in the real world.

Use of Biological Materials

Any team considering the release of a biological product must be fully aware of rules and regulations governing their country. In Scotland, EU regulation is implemented, along with further additions by both the UK and Scottish Government.

Our BabbleBlock generation process can be divided into three parts; the generation of BabbleBricks housed in acceptor plasmids, assembly of BabbleBlocks, and long-term storage of BabbleBlocks. The generation of BabbleBricks involves transformation of competent TOP10 E.coli cells. The assembly and storage of BabbleBricks and Blocks however is cell free.

The distinction between these two processes is important. The use of unnatural DNA sequences outside the lab is heavily governed. Most genetically modified organism laws however cover GMOs (genetically modified organisms) or (Genetically modified micro-organisms).

“Micro-organism: This term covers bacteria, fungi and viruses, as well as cell and tissue cultures from plants, animals or humans. Naked nucleic acid, oligonucleotides, synthetic DNA, plasmids or liposomes are not considered to be micro-organisms.” - The Genetically Modified Organisms (Contained Use) Regulations 2014. Scottish Government

In Scotland, the definition of a microorganism does not cover naked DNA. All relevant governing GM legislation in Scotland refers to GMOs and GMMs, and so exclusion of naked DNA from this allows storage of assembled BabbleBlocks outside the lab.

Our functional proof of concept follows the steps our commercialised system would have:

1. The company will make BabbleBricks

2. The company will assemble Babble Blocks

3. The user will store BabbleBricks and BabbleBlocks for use whenever needed.

4. The user will read back the data stored when needed.

Barcodes on side of tubes (Figure Left)

QR codes on underside of tubes; Can be read as a plate or individually (Figure Right)

Make BabbleBricks

As demonstrated in Proof-of-concept (link page) we have made phytobrick compatible BabbleBricks. These plasmids are the DNA component of the BabbleBrick Library. For proof-of-concept we have made X, however our 5bp coding regions allows for 1024 different BabbleBricks to exist in the library. This can be scaled up; for example, a coding region of 6 nucleotides would give 4096 different BabbleBricks. The BabbleBrick Library (and complete BabbleBlocks) would be stored in barcoded tubes for integration with an automated system for assembly, and catalogue system for storing the completed constructs.

BabbleBlock Assembly

BabbleBricks have been manually assembled into BabbleBlocks in the lab, as also seen in proof-of-concept (again link).

We can see a constructed BabbleBlock on this gel:

However, as a commercialised product we would intend to use an automated lab so that many BabbleBlocks can be generated in parallel. The script below shows an example of how we would automate all the steps.

Long-term BabbleBlock Storage

We aim to produce a solution to store long-term archival data. In the space of a summer however it is not feasible to test this. To tackle this, we have created two functional tests of storage:

1 – Short-term storage of DNA under current archival conditions.

2 – Storage of DNA under simulated long time conditions (achieved through high heat).

1 - Short-term storage of DNA under current archival conditions

The Edinburgh University Library are potential end users of our system. We discussed with them the type of environmental conditions long-term data is currently stored. We were informed that the library, like most other UK archives conform to British Standards Institution 5454 regulations. This standard is a set of both environmental guidelines such as temperature and humidity, as well as practical issues such as storage containers and fire protecting schemes. This standard was something we were unable to replicate in the lab. However the library offered to store samples for us under monitored conditions. Upon risk assessment by the School of Biological Sciences safety officer this was approved.

Part XX (a BabbleBrick housed in plasmid) was used for testing.

5μl was pipetted onto blotting paper, dried and cut out. This was placed in tube 1.

5μl was put into tube 2, and left as a liquid sample.

5μl was put in tube 3 which was left to evaporate to give a dry sample.

All three samples were placed in a labelled box, and shelved in the archives room for 2 months.

2. The above test of DNA storage under archival condition provides a measure of how easy it would be to implement our DNA storage medium into existing archival facilities. However a test that spans 2 months does not provide much information on the long-term suitability of DNA.

Data Read-back

Stored archival data is no use unless it can be read back when needed. Despite our best efforts to keep DNA stable, there is always the possibility of sequencing errors. While this will reduce with time it is still important to have mechanisms in place to combat this. Our readback software (link) has error identifying and correcting mechanisms to help minimise the event of unreadable data. This consists of checksums and optimal rectangular codes, and can be further explained here (link). To tests these mechanisms simulations were run. These input a variety of incorrect sequences, and allowed the software to correct them.

References

http://www.legislation.gov.uk/uksi/2014/1663/contents/made

http://www.legislation.gov.uk/uksi/2014/1663/contents/made

https://www.thenbs.com/PublicationIndex/Documents/Details?DocId=300479