Team:SDU-Denmark/Contribution
Contribution
As part of the PROSTATUS project, we have developed several tools, that we believe will be useful to future iGEM teams. While these contributions would allow you to quickly build powerful tools in disease detection, they can also be used to solve other problems.
This page contains a guide that helps you design your own sgRNA/Cas-based DNA or RNA detection system. The CAD-files with the designs for the PROSTATUS cup, will be made available to anyone upon request. Finally, the team has generated a model to help track the temperature of liquids in test containers, for applications that rely on heat sensitive reactions. Happy inventing!
Template-assay for testing RNA-DNA biomarkers in urine
With this general method, it is possible to quickly and cheaply screen for any specific ssRNA- or DNA biomarkers in urine.
The concept is to first select a perk or diseases you are interested in testing. Then search in the literature if there is a specific, or perhaps a combination of biomarkers expressed or not expressed in urine.
There are nine steps in the template. Each specific step correlates to the numbers in the overview banner figure "Graphical abstract"
1) When the specific biomarkers have been identified, a DNA or RNA sequence of the biomarker should be obtained. Using our java-script, it is possible to obtain a sgRNA or T7-DNA sequence for Cas13a to utilize. The sgRNA (or T7-biomarker-scaffold DNA) can be delegated to an external company for synthesis or synthesized through in-vitro transcription yourself. For protein expression of Cas13a the iGEM part contains a plasmid encoding a SUMO- and his-tagged Cas13a.
To pick the right sequence biomarker of interest
The biomarker must be secreted in urine to be detectable with this assay. It is possible to search in the literature for verification of expression of the biomarker in urine.
2) It is preferable to search in a database that has conducted a different spliced version of the gene. A recommended database is Ensembl , which supports over 50,000 genomes. If the biomarker of interest is a transcript gene, the biomarker sequence should be in an exon, which is expressed in as many splicing variants as possible. The length of the required guide sequence should be approximately 50 nucleotides.
3) When a DNA -or RNA biomarker sequence is chosen, a complimentary gRNA must be synthesized to be used as a guide for Cas13a. A T7 promoter sequence can be added to the initial DNA-sequence to utilize T7 polymerase to produce the finished sgRNA.
The sgRNA for Cas13a is composed of a scaffold part and a spacer part. The appropriate construct is: 5' - Scaffold + Spacer – 3
The scaffold-sequnce for Cas13a is: 5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAU – ‘3
The spacer is the target, which is a reverse complementary sequence from a given part of the biomarker-sequence of interest. The spacer sequence should be 28-31 nucleotides
The iGEM team from Rochester 2020 has produced an excellent biomarker database, where it is possible to find relevant biomarkers for different diseases.
Express Cas13a protein for use
4) It is possible to order a full plasmid ready for transformation into a bacterium for expression. The plasmid should contain an antibiotic resistance gene, restriction sites to insert genes into the plasmid (e.g. EcoRI, XbaI, SpeI, and PstI) and an origin of replication. It is highly recommended to choose a backbone from iGEM, which already includes all the necessary parts e.g. pSB1c3 (conventional backbone).
After choosing the appropriate backbone, both the Cas13a gene and backbone-plasmid should de digested so that they are ready for ligation. The Cas13a gene is available as the iGEM part: BBa_K3602020.
After ligation, the plasmid (w. Cas13a ) is ready for tranformation into an E. coli strain, which has good properties for plasmid amplification and fast generation time (e.g. E. coli top 10 ).
5) Isolate and purify the amplified plasmids, and transform them into a strain that has the properties to make large quantities of protein upon induction (e.g., E. coli BL21).
6) Induce the strain with the plasmid and isolated the Cas13a protein with appropriate protein purification method (e.g. for Immobilized metal affinity chromatography with the matrix Ni-NTA Agarose). Store the purified Cas13a in an appropriate store-buffer (e.g. 30-50% glycerol) and freeze (-20°C to -80°C).
Incubation and amplification
If the concentration of the biomarker in urine is expected to be found in a low concentration, it would be necessary to amplify the present biomarkers, to decrease the prevalence of false negatives. It is recommended widely to use a recombinase polymerase amplification (RPA) reaction, as it can be used outside of laboratories and is a “one-pot” isothermal amplification reaction. The RPA reaction is further described in the laboratory section and in this article about rapid detection of virus . It is also possible to add the T7 polymerase to the reaction to transcribe a DNA-biomarker to ssRNA for detection by this assay.
7) The next step is to test whether the biomarker of interest is present in the given urine sample. This is simply done by first incubating the stored Cas13a protein and sgRNA for 10 minutes at 37°C, followed by incubation of the Cas13a-sgRNA-complex together with the urine. We have developed a ready to use 3D-printed cup that is able to carry out the detection of four different biomarkers at once. A further in-depth description of the PMT-cup is available at the hardware page . We have created a mathematical model that can show the reaction temperature over time, to ensure optimal temperature during the reaction when using the PMT-cup at the modeling page .
When using our flow strips for detection, it is crucial to inhibit all the naturally occuring RNases in urine, as these cleave the RNA reporters thus give false positive results. The strong properties of proteinase K to digest other proteins, including RNases, makes proteinase K great for rapidly inactivate nucleases, ensuring correct test results. After successful inhibition of RNases, Proteinase K itself must be inhibited as to not degrade the Cas13a protein. This is done by adding PMSF.
A protocol for inhibition of nucleases and later proteinase K is greatly described by Promega ‘Proteinase K’ or Inactivating of RNase in urine and spit
8) Our 3D-model illustrated in an automated video
The specific CAD-files of the prototypes and related accesories are openly available to download from here.
9) Flow-strip detection assay
To verify the presence of the given biomarker in urine, a universal Lateral Flow Assay can be utilized. This assay contains RNA-reporters, which are cleaved when the process of collateral cleavage is activated in Cas13a. The RNA-reporter sequence consists of a random RNA sequence, where biotin is bound to the one end (( https://www.milenia-biotec.com/en/product/hybridetect/ )) and a FAM-particle to the other end, which becomes bound by the gold particle, allowing for readout. The biotin has an affinity for the first part of the flow-strip, as it contains a biotin receptor. When the RNA-reporter is not cleaved, the biotin binds to the control line on the strip, giving a negative readout. However, when the RNA-reporter is cleaved, the gold particle becomes ‘free’ and it is possible for it to bind to anti-FAM-antibodies at the test line. The universal Lateral Flow Assay is available here .
Contribution conclusion
Our hope with this template contribution, that future iGEM teams and other people interested in developing a biomarker test, can do this with the help of synthetic biology. Our hope is, that this template gives them a head start, and allows them to avoid the problems and pitfalls we have met so far. Thus, future teams can stand on our shoulders and hopefully achieve more and possibly reach a place of testing on real urine. A point we ourselves came close to.
