“Cercival Cancer Screening Kit”
A. DETECTOR
1. Background
Cervical cancer is one of the most common cancers in womenworldwide,with an estimated global incidence of 470,000 newcases and approximately 233,000 deaths per year. Then, we try to make an idea to build a new prototype for early detection to cervical cancer, especially caused by HPV 18 and HPV 16. From the previous results of the two teams, Calgary Team 2013 and Freiburg Team 2012, we build a new prototype with some combination from those teams. We learn about the prototype sensor from Calgary Team 2012 that consist of detector, linker, and reporter that combined into one small test pack. Then, to make the detector, we learn from Freiburg Team 2012 to constructing a new specific TALEs that can bind to our target sequences and we called it TALE 1 and TALE 2.
TALEs or Transcription Activator-Like Effectors comes from natural bacterial effector proteins used by Xanthomonas sp. to modulate gene transcription in host plants to facilitate bacterial colonization (Boch J and Bogdanove, 2010).The central region of theprotein contains tandem repeats of 34 amino acids sequences (termed monomers) that arerequired for DNA recognition and binding (Kay, 2009). Here is the natural structure of TALE from Xanthomonas sp. :
Figure 1. Structure of TALEs derived from Xanthomonas sp. (Sanjana et al, 2013)
2. What did We Do ?
As explained before in the Background, we try to make a prototype based on the concept of Calgary Team 2013. So we construct a new TALEs that can bind with our target sequences, especially in HPV 18 and HPV 16. For building the detector, we received help from Freiburg Team 2012 to construct two new TALEs. First, we search the target sequences with the alignment of whole genome of HPV 18 and HPV 16 via NCBI and MEGA5. We search two target sequences in left side and right side. Here are some works that we’ve done:
Left side: site 7106 until 7119
Right side: site 7851 until 7864
3. CONSTRUCTING TALEs
Based on Freiburg Team 2012 method, we designed our TALE into 2, TALE 1 and TALE 2.
For TALE 1, we use:
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(1) BBa_K747012,
(2) BBa_K747016,
(3) BBa_K747042,
(4) BBa_K747048,
(5) BBa_K747064,
(6) BBa_K747091
For TALE 2, we use:
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(1) BBa_K747012,
(2) BBa_K747026,
(3) BBa_K747036,
(4) BBa_K747052,
(5) BBa_K747076,
(6) BBa_K747095
4. VECTOR
For the vector for our TALEs, we received help from Lucas Schneider (member of Freiburg Team 2012) and Nicole Densch (Labmanager from BIOSS, Freiburg). We request the pTALEN and pTAL – TF from BIOSS and sent it to us on Whatman paper. Based on Sanjana et al. (2013), TALE-TFs are constructed by replacing the natural activation domain within the TALE C-term with the synthetic transcription activation domain VP64.
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Figure 2. TALEs can be used to generate custom transcription factors (TALE-TFs) and modulate the transcription of endogenous genes from the genome (Sanjana et al., 2013)
TALENs are constructed by fusing a C-term truncation (+63aa) of the TALE DNA binding domain with the non-specific FokI endonuclease catalytic domain. TALENs form dimers through binding to two target sequences separated by ~17 bases (Sanjana et al., 2013).
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Figure 3. TALE nucleases (TALENs) can be used to generate site-specific double strand breaks to facilitate genome editing through non-homologous repair or homology-directed repair (Sanjana et al., 2013)
B. LINKER
As we’ve said before that we inspired by the Calgary Team 2013’s project, so we decided to learn the linker from previous work by them. The E/K coil is a heterodimeric coiled-coil that often used for biosensors and as an expression and affinity purification tag. The E coil contains all glutamic acid residues and the K coil contains all lysine residues. This coiled-coil can be found in a wide variety of proteins and contain a single type of secondary structure or the α-helix. There are two ways to increasing the stability of this sequence: the hydrophobic core of the coiled-coil (positions at a and d) and the α-helical propensity of surface esposed positions at b, c, e, f, and g (Litowski, 2002).
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Figure 4. Helical wheel of the E4/K4 heterodimer (Litowski, 2002)
We used linker to connect Tale and reporter. We used E and K coil since they can interact each other with high specificity. E-coil was fused to amilCP blue chromoprotein (reporter), while K-coil bound to Tale. This fusion make the reporter can be linked to Tale (detector) through K coil-. This fusion involving PCR and these part was digested and ligated as Igem protocol.
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C. REPORTER
In order to make sure the presence of DNA from HPV 16 and 18, we need reporter which connect to detector. Fluorescent protein usually used as reporter gene to study about gene expression. Gene encoding fluorescent protein was synthesized in four stages (Laufer et al, 2013). Chromoprotein can be used as good reporter. Another useful feature of the chromoproteins is the “kindling” behavior, which makes them prospective photoactivatable markers. In this project, we used amilCP blue chromoprotein. This chromoprotein was extracted from Acropora millepora and display purple-blue colour (Alieva et al, 2008).
This is one of chromoprotein collection from Uppsala Team 2012. This chromoprotein will be connected to detector using linker. amilCP bind to E coil and interact with Tale-K coil. This reporter show the presence of Human Papilloma Virus by colour change. When the HPV DNA is bind to detector, spesific sequence of HPV will be recognized and blue colour appears in the second strip. The Colour intensity will be detected in our software and we can determined the risk level of cervical cancer disease. Dark blue colour show the amount of DNA from HPV 16 and 18 is high and indicate the patient get severe cervical cancer.
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Figure 6. amilCP with his tag and C-terminus E coil
D. PROTOTYPE
With those concept as explained before, we’re trying to make a prototype for early detection to cervical cancer that has some common with test pack. These are our model for the prototype:
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We test the sample from menstrual blood that we hypothesized if she got cervical cancer, the menstrual blood will go through the endometrium and then to cervix which is can bring some of the infected cells. Then the give a little bit of menstrual blood to the applicator. Inside the test pack we’ll use nitrocellulose membrane to get the result. Next steps is the binding from TALE 1 that already bind with the linker and reporter to the HPV DNA that can be found in menstrual blood. We can’t be sure if the TALE 1 and HPV DNA is binding without the TALE 2, so the sample will be catch by TALE 2 that stands for positive line. If that two TALEs can’t bind to the DNA it means negative result. Meanwhile we make control line too to compare the positive line and the control line.
E. SOFTWARE
This software help user to early detect her cervical cancer with capture image from c3+ kit. This software also inform all about cervical cancer, especially preventing, screening, and therapy.
REFERENCES:
- Alieva, N.et al. 2008. Diversity and Evolution of Coral Fluorescent Proteins. (http://plosone.org)
- Kay S, Hahn S, Marois E, Wieduwild R, Bonas U. 2009. Detailed analysis of the DNA recognition motifs of the Xanthomonas type III effectors AvrBs3 and AvrBs3Deltarep16. Plant J. 59:859–871.
- Boch J, Bonas U. 2010. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol; 48:419–436.
- Litowski, Jennifer R. And Robert S. 2002. Hodges. Stability, and specificity propensity on protein folding, -helical α hydrophobicity and -helical coiled-coils: effects of α designing eterodimeric two-stranded. J. Biol. Chem, 277:37272-37279.
- Sanjana et al. A Transcription Activator-Like Effector (TALE) Toolbox Genome Engineering. Nat Protoc.; 7(1): 171-192.
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