Project Inspiration and Description
Most of our team members are biology students who have to spend some time on practical courses in the fields as a part of their curriculum. It is not uncommon to take off ticks at the end of the day. However, the detection of tick-borne diseases in the field remains an outstanding issue. The market products available nowadays imply to send the tick in the laboratory for further investigation, but it is a solution unsuitable for barely accessible regions.
Each year around 500,000 Lyme disease cases caused by tick-borne Borrelia spp. are reported worldwide. Prompt detection of Borrelia infection is crucial for effective treatment. Quick point-of-care detection of the pathogens in an extracted tick is therefore important. To solve this problem, our team proposes a portable biosensor device that can detect the presence of Borrelia spp. in ticks - LymeExpess. It comprises tick homogenization followed by the detection of pathogen-specific DNA motifs. The detection is based on using specially engineered dCas proteins from various organisms fused with split domains of β-lactamase. The dCas complexes target the complementing split domains to the nearby DNA locus allowing for the fully functional reporter protein to be formed. The products of the colorimetric reaction catalyzed by the protein are detected with an embedded spectrophotometer. This yields an easy to use, cost-competitive and quick testing device that can be used even in field conditions.
Lyme Borreliosis is the most common tick-borne disease in both North America and northern Eurasia (Jones, 2009; Nelson et al., 2015). The risk areas coincide with the habitats of the vectors, ticks of Ixodes ricinus species complex (Stanek et al., 2012)⁠.
figure 1
Figure 1. Global distribution of the vectors (Ixodes ricinus species complex) of Lyme borrelia (Stanek et al., 2012)
Annually about 500 000 people come to the Center for Hygiene and Epidemiology with tick bite only in Russia (fig. 2). However, the probability of bacteria transmission from tick to human varies and determined by tick species, location, and length of feeding. According to the CDC, the tick must be attached for 36 to 48 hours or more before the Lyme disease bacterium can be transmitted
figure 2
Figure 2. Reported tick bite cases by year, Russia, 2010-2019 (Federal Service for Supervision of Consumer Rights Protection and Human Well-Being)
The percentage of ticks carrying highly depends on the location of the bite. In Russia, Borrelia spp. was detected in 14,4% of tested ticks in 2012 (Federal Service for Supervision of Consumer Rights Protection and Human Well-Being). The distribution of risk zones inside the country forms a belt in the south (fig. 3).
figure 3
Figure 3. The long-term average incidence of Lyme borreliosis in the districts of Russia, 2010-2017 (Federal Service for Supervision of Consumer Rights Protection and Human Well-Being)
The same level of infected ticks in the US is shown by a study published in 2018 with the highest prevalence of B. burgdorferi in black-legged tick, I. scapularis - the main vector in the northeastern US states (fig. 1) (Nelson et al., 2015)⁠. The percentage of bacteria vectors ranges from 6.5–23.1%. Moreover, comparing tick distribution maps in the US the spread of infected ticks from coastal to central areas can be observed (fig. 4).
figure 4
Figure 4. Distribution of reported Lyme disease cases in 2012, 2015 and 2017, US (CDC). 1 blue dot placed randomly within the country of residence for each confirmed case
6 500 cases of Lyme borreliosis were diagnosed in Russia last year. Fortunately, the number of cases did not increase in the recent decade (fig. 5, A). But in such countries of Europe as the Czech Republic, Poland, and Slovenia the number of cases diagnosed every year was raised in 1996-2010 (fig. 5, B-D). The same situation can be observed in the US (fig. 5, E).
To compare a number of cases incidence is used, the number of new cases occurring within a period (e.g., per month, per year). The incidence rate is reported as a fraction of the population at risk of developing the disease (e.g., per 100,000 or million population).
The incidence in the northeastern countries of Europe also increased during 1996-2010 (fig. 6, B-D. Thus, in Europe, the rise in the number of cases is connected with the increase in percentage. Whereas, the increase in the number of cases in the US is explained by the simple natural increase of the population (fig. 5).
The recent studies conducted by CDC shows the actual number of people diagnosed with Lyme borreliosis is ten times as large as reported every year by physicians and reached 400 thousand of infected people only in the US (Hinckley et al., 2014; Nelson et al., 2015).⁠⁠
figure 5
Figure 5. Reported cases of Lyme disease by year in Russia, 2010-2018 (Federal Service for Supervision of Consumer Rights Protection and Human Well-Being), Czech Republic, 1998-2008, Poland, 1998-2010 (CISID WHO) and the US, 2007-2017 (CDC)
figure 6
Figure 6. Incidence of Lyme disease by year in Russia, 2010-2018 (Federal Service for Supervision of Consumer Rights Protection and Human Well-Being), Czech Republic, 1998-2008, Poland, 1998-2010 (CISID WHO) and the US, 2007-2017 (CDC). References
  1. Hinckley, A. F., Connally, N. P., Meek, J. I., Johnson, B. J., Kemperman, M. M., Feldman, K. A., … Mead, P. S. (2014). Lyme disease testing by large commercial laboratories in the United States. Clinical Infectious Diseases, 59(5), 676–681.
  2. Jones, P. (2009). Public Health Significance of Urban Pests. Nursing Standard, 23(24), 31–31.
  3. Nelson, C. A., Saha, S., Kugeler, K. J., Delorey, M. J., Shankar, M. B., Hinckley, A. F., & Mead, P. S. (2015). Incidence of clinician-diagnosed Lyme disease, United States, 2005–2010. Emerging Infectious Diseases, 21(9), 1625–1631.
  4. Stanek, G., Wormser, G. P., Gray, J., & Strle, F. (2012). Seminar Lyme borreliosis. Seminar, 379(February 2012), 461–473.
Wet lab
In order to create a working molecular system - the molecular basis of the biosensor - as well as to test and describe its functioning, we performed a series of ‘wet’ laboratory experiments. We focused on working with split β-lactamase fused with dCas9 from Streptococcus pyogenes.
During the first part of our work, we had to choose the split reporter protein to create a fusion system with dCas9 proteins. β-lactamase with His-tag and various luciferases. The plasmids were expressed in DH5α cells. The results of purification of the plasmids are presented below (fig.1):
Figure 1
Figure 1. Agaroze gel electrophoresis. Purification of plasmid DNA from DH5a cells. M - GeneRuler 1 kb DNA Ladder, beta-lac - Part: BBa_K1189009, Vibrio luc - Part: BBa_K325909, Renilla luc - Part: BBa_J52008)
Subsequently, a plasmid containing β-lactamase with His-tag under IPTG-inducible promoter (Part: BBa_K1189007, fig.2) was cloned into both BL21 De3 cells and DH5a. Enzyme activity was analyzed by growth curves in the presence of the antibiotic ampicillin. The results of these experiments are presented in the biobricks section.
After obtaining data on the activity of lactase using the test in the presence of antibiotics, we proceeded to the creation of molecular constructs for our future test system (biosensor). The development of this ampicillin test was necessary for us, since we decided to test the work of our system in the cell.
Figure 2
Figure 2. plasmid purification, agarose gel. M- GeneRuler 1 kb DNA Ladder
We decided to use the following constructs as a source of dCas9 protein constructs (fig.3):
Figure 3
Figure 3. plasmid purification. agarose gel electrophoresis
M - GeneRuler 1 kb DNA Ladder
To confirm the presence of the required biobrick in the plasmid, we performed a restriction analysis, as well as PCR using standard VF2 and VR primers (fig. 4)
Figure 4
Figure 4. Agarose gel electrophoresis. M - GeneRuler 1 kb DNA Ladder. 1-3 - PCR fragments containing the insert Nlac_dCas; 4-5 PCR fragments containing the insert. Clac_dCas
As a result of restriction analysis and PCR, we were able to confirm the presence of the insert of 4000 bp. Then we had to confirm the sequence of Clac_dCas9 as it was inconsistent and Nlac_dCas9. Sequencing characterized the target genes without mutations and deletions (fig. 5)
Figure 5
Figure 5. The results of Clac_dCas9 sequence added to the registry
We decided to add the T7 promoter and the double terminator to Clac_dCas9. As a promoter we selected a well-described biobrick promoter; as a double terminator, we selected two possible terminators (Part:BBa_B0017 = p34e, Part:BBa_B0015 = p35c, Part:BBa_K525998 = p13m)
We also obtained the necessary fragments from the igem plates (fig.6).
Figure 6
Figure 6. Agarose gel electrophoresis. M - GeneRuler 1 kb DNA Ladder.
Now we are assembling the constructs containing all the necessary elements to obtain stable expression of cas proteins.
Obtaining different variants of CRISPR/Cas systems for the biosensor improvement
Based on the results from the modeling part, our Team aimed to obtain a wide spectra of CRIPSR/Cas systems to use them for making the fusion construct with β-lactamase. We received from different sources the plasmids carrying native and catalitically dead versions of Staphylococcus aureus, Streptococcus thermophilus, Campylobacter jejuni and Proteobacteria phylum. The plasmids were transformed into host strains (DH5a and XL10) and plasmid DNA was purified for the further molecular cloning to the β-lactamase split domains.
Further steps we will definitely make next year :
  • Purification of split proteins and verification of their functionality
  • Evaluation of reconstitutional split systems and its testing with target sequences (gel shift)
  • Testing the molecular system with control samples
1. Molecular Modeling
To detect Borrelia DNA we utilized parts of split β-lactamase fused to N- and C-termini of Cas proteins using glycine-serine linkers (GGGGS). Binding to Borrelia genomic DNA causes the parts of the split system to reunite. Thus, it is necessary to ensure that the distance between the parts of the split system is enough for the linkers to reunite the complex. At the same time, when two Cas proteins are too close, they sterically hinder the complex assembly. To determine the optimal distance between two Cas proteins, we performed molecular modeling in the Chimera program.
Project details
Based on the experimental data of the team participating in iGEM Peking 2015, we chose the PAM-out orientation (see Fig. 1) for our system of two Cas proteins. The N-part of β-lactamase is connected to the C-terminus of the Cas protein (PAM on Crick strand 3 ’-> 5’); the C-part of β-lactamase binds to the N-terminus of the Cas protein (PAM on Watson strand 5 ’-> 3’). The modeling aimed to determine the optimal distance between two Cas proteins so that the N-terminus of one Cas protein and the C-terminus of the other one are as close to each other as possible.
figure 1
Fig. 1. Possible orientation of Cas proteins (PAM-in and PAM-out) upon binding to DNA.
To design these systems we used Cas9 proteins isolated from different bacterial species (see Table 1). These Cas proteins differ in their structure as well as in the length and sequence of PAM. Using a Cas protein with a longer PAM is preferable as it increases the specificity of target recognition compared with a Cas protein with a shorter PAM. Furthermore, N- and C-parts of β-lactamases fused to different types of proteins increase the sensitivity of the system.
Cas Organism PAM length of target PDB id
Sp Streptococcus pyogenes NGG 20 6O0x
Sa Staphylococcus aureus NNGRRT/NNGRR(N) 21/22 5CZZ
Cj Campylobacter jejuni NNNNAYAC 22-24 5X2G
St Streptococcus thermophilus NNAGAAW 20 None
Table 1. Cas proteins used in modeling.
The results of modeling are shown in Table 2.
1 Cas 2 Cas Length of spacer
(between 2 Cas)
Sp Sp 22
Sp Sa 20-21
Sa sa 20
Table 2. The results of determining the spacer length based on modeling.
Figure 2 shows a model of two SpCas9 proteins binding to DNA; the N-terminus of the Cas protein is highlighted in red, the C-terminus of the Cas protein is in green. The model shows the minimum possible distance between N and C termini.
figure 1
Fig. 2. The model of two SpCas9 proteins binding to DNA.
  • The results of modeling will be tested experimentally.
  • We plan to develop models with StCas9 and CjCas9 proteins.
2. Molecular Dynamic
The main idea of confirming the mobility of linkers and the high probability of the joining of β-lactamase into an active state, was a plan to carry out molecular dynamics in the GROMACs package. We have decided to use the 6o0x.pdb file from PDB database as it had the RNA is its crystal structure. Once we have started to create our system, big structural problem appeared. Almost 40% of structure was missing, which is inappropriate to start the molecular dynamics. First of all, we have tried to fill in the missing parts using 1) Swiss-Model and 2) Modeller program for Chimera. Unfortunately, both methods could not provide proper results, because there were not enough different structures, on which the analysis could be based.
Right now we are trying to create a proper system without missing structure.
3. Kinetics
Please click on the link below which will redirect you to to see our code, our model and its description (you will see our github repository, everything is in a file called KinProj_Final.ipynb).
Warning: If you can't see the plot (when you are viewing the file mentioned above), click on the first text field (with the code) and press the "Run" button above.
Variable Annotation Type Value Source
[sgRNA/dCas-Blac]1/2 Concentration of each of the sgRNA/dCas-Blac Parameter
[DNA] Target DNA concentration Parameter
[S] Substrate (nitrocefin) concentration Parameter
[P] Product (hydrolyzed nitrocefin) concentration
OD = 0.35
Known OD = 0.35 From Nickolay Kristovskiy
ε Extinction coefficient for hydrolyzed nitrocefin Known ~20,500 M-1 cm-1
A Activity of enzymatic system (complemented β-lactamase) Known
Km Km of enzymatic system (complemented β-lactamase) Known 10 uM “Kinetic Properties of Four Plasmid-Mediated AmpC β-Lactamases”
Bauvois et al, Antimicrobial Agents and Chemotherapy Sep 2005, 49 (10) 4240-4246
Kd1/2 Dissociation constant for each of sgRNA/dCas complexes Known 0.5 nM “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9”
Sternberg et al, Nature volume 507, pages 62–67 (06 March 2014)
1.5 nM “Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage”
Cencic R, Miura H, Malina A, Robert F, Ethier S, Schmeing TM, et al. (2014) PLoS ONE 9(10): e109213
>0.17 nM
<100 nM
“Programmable DNA looping using engineered bivalent dCas9 complexes”
Nan Hao, Keith E. Shearwin & Ian B. Dodd Nature Communications volume 8, Article number: 1628 (2017)
t Time Parameter
Design of guide RNA
To design guide RNA to detect Borrelia Spp. we used 4 genomes from NCBI Genes: Borreliella burgdorferi, Borreliella bavariensis, Borreliella afzelii, Borreliella garinii. We searched targets in DNA by python code with optimal length of spacer (distance between 2 CRISPR/Cas systems) from Molecular modelling. To search all possible targets we varied length of spacer (+- 10 bp) and threshold of pairwise matching of targets among different genomes.
The short list of targets contains 10 sequences (threshold 20/20), 5 of them are located twice in genomes, in duplicated gene 23S RNA. Other targets are located in genes, which codes: UDP-glucose pyrophosphorylasе (pseudogene), purine-binding chemotaxis protein, 16S ribosomal RNA, rod shape-determining protein MreC, penicillin-binding protein. The extended list of targets (threshold 19/20) contains 65 targets. Also we checked potential targets in tick genome Ixodes scapularis (with threshold 16/20) and its microbiote (this part was done during collaboration with Team NU Kazakhstan).
Length of spacer Threshold Number of targets
21 20 4
22 20 10
23 20 9
As devices, detecting a chromogenic response, we have developed photometers and spectrophotometers. Their distinctive feature is the lack of optical lenses, for the sake of mobility and reliability. We calibrated all our instruments by comparing their data with the data of the ultrospec 1100 pro spectrophotometer from Biochrome.
LymePhoton-M is a photometer, that detects the optical density of solutions at certain wavelengths (470 nm, 590 nm, 630 nm). It is a system of LEDs and a photodetector connected to a microcontroller (esp 8266). The cuvette holder has two positions. In the first position, the light from the led goes straight to the photo sensor. In the second position, it will be possible to detect the fluorescence of biometrics, since the light from the led will not strike the photodiode directly.
Figure 1. Appearance of the device LymePhoton-L.
Figure 2. Electrical diagram of LymePhoton-M
Quantity Components Cost Final
1 OPT101P $0,82 $0,82
1 Esp 8266 R1 D1 $1,95 $1,95
1 BL-L522UBC $0,25 $0,25
1 BL-L522UYC $0,11 $0,11
1 BL-L522UEC $0,11 $0,11
2% Resistors (300 pieces) $1,38 >$0,00
5% Wire (10m) $2,34 $0,12
12% PLA black plastic (1kg) $12,19 $1,46
13% Matrix board $1,36 $0,18
Total - $4,97
Table 1. Components used for the construction of LymePhoton-M
LymePhoton-S — is a mini-photometer with minimal functionality. Packed in a small case, with only one led, one brightness level, a small cuvette holder, photodetector and a microcontroller (esp 8266).
Quantity Components Cost Final
1 OPT101P $0,82 $0,82
1 Esp 8266 R1 D1 $1,95 $1,95
1 BL-L522UBC $0,25 $0,25
1% Resistors (300 pieces) $1,38 >$0,00
5% Wire (10m) $2,34 $0,12
5% PLA black plastic (1kg) $12,19 $0,61
13% Matrix board $1,36 $0,18
Total - $3,93
Table 2. Components used for the construction of LymePhoton-S
We have posted on our page in Github the latest versions of the code for our devices and also cases files.

MSU Biological Faculty
Sechenov University
Chroma Technology
Twist Bioscience
Integrated DNA Technologies
New England BioLabs
Unstoppable Technologies
BioSchool Piligrim
institute of fundamental medicine and biology