Project Description

Inspiration

Our Inspiration came from three different directions

First of all and most importantly we were determined to solve the problems mentioned earlier and there is hardly a better or stronger inspiration than the urge to solve a global problem. We knew that our solution would lie in the field of synthetic biology. This inspired us to design a novel fusion protein. It should be able to recognize any DNA sequence of interest and be able to give a visual readout if the sequence of interest was present in the sample. We knew from the beginning that we wanted to make use of the unique abilities of dCas9. But we had to decide what to fuse it to and how to design our construct. Read more about the whole process here.

Secondly we were inspired by a paper that found the way to us via the old college of our teammate Sebastian, Dr. Paul Szekely. [7] He was one of the first stakeholders that we got in contact with. He had been working with Sebastian in the amazonian rainforest identifying different species of animals by PCR and was intrigued by our idea to test for any sequence of interest with simple and cheap means, right there on the spot without having to move to the laboratory. He ensured us how very much needed and helpful such a device would be for is work and recommended the paper “Nucleic acid extraction within 30 seconds” to us. With that he sent us back to the drawing board. The possibility to extract DNA with cellulose paper in such a short amount of time without the need for laboratory equipment shifted our whole project idea. This also solved the problem of how to make sure that we only get a readout when the DNA sequence of interest is present.

Our third inspiration was the project of the team of Delft from 2017 [8] which had a beautiful and inspiring project about a tool to test for the presence of antibiotic resistance in a certain microbe infecting the udders of cows. They used a variant of Cas9, Cas13a, which is able to bind to RNA instead of DNA. They did not only show that it is possible to design and build such a project in the short amount of time available to an iGEM team but they also demonstrated that a Cas protein could work in the field, literally, since their method was applied by farmers to test their infected cows.

Description

Our method consists in a short and easy DNA extraction in which the DNA is bound on a cellulose paper strip. Bound to the strip the DNA is transferred into a solution containing our novel fusion protein: dCas9 linked to horseradish peroxidase (HRP). The dCas9-HRP, will only bind to the immobilized DNA if the sequence of interest is present. Upon transferring the paper strip into the last vile containing a substrate (TMB) for HRP. Finally, a color reaction will only be induced if the fusion protein could bind the specified DNA in the previous step.

The readout will be shown on the paper by the development of a deep blue color, indicating that the sequence of interest is present. One can imagine this similar to the color change of a pH strip used to test the acidity of a solution.

To understand this method in more detail, please read-on:

In the first step, DNA will be extracted directly from a raw cell lysate of either microbial cells or human samples, taken by buccal swab, by immobilising it on a cellulose paper (based on a method developed by Zou et al. 2017). [9]

After a short washing step (Figure 1) to remove proteins and membrane particles from the cell lysate, highly pure DNA with a concentration of around 40 ng/µl will be left on the paper (OD260/280 ~ 1.8). Check out our results here.

In the second step, the cellulose paper is dipped into a solution that contains dCas9 linked to HRP. The dCas9 will recognize and bind to the specific DNA sequence that is determined by its guideRNA.

Figure 1 - Basic procedure of the DipGene gene detection process.
The DNA from the sample is bound to a cellulose paper and the presence of a specific sequence of interest
is indicated by a color readout visible to the naked eye.

In the third and last step, the strip will be transferred to a solution with HRP substrate. When the fusion protein dCas9-HRP has bound to the DNA of interest, it will be transferred to the third solution where a colour change of the HRP substrate will be triggered. The colour change will only occur when the sequence of interest is present.

How the new RCF25 standard will be used to assemble our fusion protein is described in detail here. Cas9 and the reporter, HRP, will be designed by mutating all restriction enzyme sites interfering with iGEM standards. Prefix and suffix of the RCF25 standard will flank our constructs. This will allow an easy and flexible reshuffling of the genes of interest to arrange fusion proteins in any desired order. Therefore, we also designed a Maltose Binding Protein (MBP) and a linker as such and used them to assemble the final construct, which will additionally carry a strep-tag for purification.

The construct will look like this:

The final construct consists of three novel BioBricks designed by our team as well as the HRP BioBrick of Georgia State team 2015 and the strep-tag designed by the LMU-Munich team of 2012.

For the future development of our diagnostic technique, the basic idea of the BioBrick assembly comes in handy. This will enable us to use the dCas9 construct, flanked by the Biobrick prefix and suffix, and attach different reporter enzymes like alkaline phosphatase or glucose oxidase. All our BioBricks are designed in a way that makes their recombination into different constructs convenient, easy and fast. [10] With the different reporter enzymes mentioned, it will be possible to get a readout in different colours. With each reporter catalysing a colour reaction for a different substrate, one could test for several different disease genes at once. For example, for the disposition for breast cancer and Alzheimer’s or the integration of genes of the HI-virus in the human genome (Figure 2).

For laboratory applications, it could additionally be of interest to fuse reporters to a variety of Cas-proteins that can be used to target either RNA or DNA, according to the need. This opens the possibility for several applications in the laboratory such as targeting mRNA to study the human or microbial transcriptome to monitor gene expression levels. The method can also be used to genotype model organisms or screen bacteria for successful integration of a genetic constructs without having to go through all the time consuming steps of DNA extraction, PCR and gel electrophoresis. Due to the easy adaptability of this process to target any sequence of interest, only limited to the imagination of designing the guideRNA, the applications are virtually unlimited.

Figure 2 - A potential future extension of the project, in which one single
test can give readouts in different colors for several disease genes.

References

[1] https://www.who.int/genomics/public/geneticdiseases/en/index2.html

[2] https://www.genome.gov/about-genomics/policy-issues/Privacy

[3] https://www.who.int/genomics/elsi/gentesting/en/

[4] Melissa Gymrek, Amy L. McGuire, David Golan, Eran Halperin, Yaniv Erlich, Identifying Personal Genomes by Surname Inference, Science 18 Jan 2013

[5] Emilia Niemiec, Heidi Carmen Howard,Ethical issues in consumer genome sequencing: Use of consumers' samples and data, Appl Transl Genom. 2016 Mar; 8: 23–30

[6] https://2017.igem.org/Team:TUDelft

[7] Zou, Y., Mason, M. G., Wang, Y., Wee, E., Turni, C., Blackall, P. J., Traut, M., Botella, J. R. (2017) Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLoS biology, 15(11): e2003916.

[8] New England Biolabs (2019) BioBrick® Assembly. Retrieverd from https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/biobrick-assembly [08.06.2019]