“A BETTER FUTURE WITH SYNTHETIC BIOLOGY”

Study of synthetic biology emerged as a multidisciplinary science that help people in solving various problems. Among the general public, synthetic biology is famous as bioengineering and extreme genetic engineering which have an advantage in redesigning a life through genetical circuit, thus allows people to manipulate a new organism for use in treatment and diagnosis of disease, provision of quality seeds, remediation of environmental pollution caused by industrial and mining as well as the mitigation of global warming.

Indonesia as one of the currently high development countries in science is allowing for synthetic biology to grow rapidly. Prospects for the synthetic biology's development in Indonesia, among others, is the independence of technology in the health sector include the development of a specific diagnosis, the provision of pharmaceutical raw materials, and technology screening for Indonesia natural resources which have potency to treat various types of diseases.

Brawijaya University iGEM team will develop synthetic biology in order to solve health sector problems, focus in Cervical Cancer. We have three sub-projects, they are: plant engineering for cervical cancer prevention, developing Cervical Cancer Care Program application for smartphone as helpdesk, and scanning technology for natural materials treatment (SCT Kit).

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“Prevent is Better than Cure”

Indonesia is an agricultural country that most of their society works as farmer. One of commodity in Indonesia is tea plantation which is located in Malang (Brawijaya University takes place). Malang is resited at 440-667 (mdpl) altitude, one of the tourist destinations in East Java because of the potential of natural and climatic owned. Malang climate conditions during 2008 temperatures recorded ranging from 22.7 ° C to 25.1 ° C. While the maximum temperature reached 32.7 ° C and minimum temperature of 18.4 ° C. The average air humidity range 79% - 86%. With a maximum moisture content of 99% and a minimum at 40%. These conditions favor the development of the tea plant (Camellia sinensis).

Tea extract contain EGCG used as the most effective antioxidant to against cervical cancer. But, levels of EGCG (Epigallocathecin gallate) in tea is very slightly. So, we will increase the level of this EGCG compound using siRNA to knockdown gene non-EGCG with expectation it will produce high yield of antioxidant. The applications are for supplement or feminine hygiene.

Tea plant (Camelia sinensis) has catechin compounds that contains antioxidants. Catechin family that most effective used as antioxidant is Epigallocathecin gallate (EGCG). Based on research in vitro or in silico, EGCG is able to inhibit the proliferation of cancer cells because stop over-expression between L1 HPV 16- EGFR (Epithel Growth Factor Receptor) bond but there are few of EGCG content in tea plant. Based on these problems, we would to over- expression EGCG by knockdown non-compound EGCG gene, so hopefully the content of EGCG on tea are more prominent. We design siRNA sequence from LAR gene (to inhibit non-EGCG compound) using bioinformatics tool and use P97 promoter to initiate expression in mammalian cell (using HeLa cell). That part will be insert into the psB1C3 linear backbone. To checking performance of this part, we insert sequence target of siRNA into the plasmid Bba_E0240 and get the promoter CMV from Bba_K747096 part. We also prepare for positive control and negative control for this experiment. Positive control part containing siRNA for GFP, insert into Bba_K747096 part that contain CMV promoter, and negative control containing CMV promoter and insert to the Bba_E0240 part containing GFP. Each part will be growth in HeLa cell culture and observed the succeding by fluorescent microscope.

“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:

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:

(1) BBa_K747012,
(2) BBa_K747016,
(3) BBa_K747042,
(4) BBa_K747048,
(5) BBa_K747064,
(6) BBa_K747091
For TALE 2, we use:

(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.

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).

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).

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.

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.

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:

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.

“SCT - Screening Cancer Therapy”

SCT (screening Cancer Therapy) is a kit which contains of hela cells as cervical cancer cells and it was designed to screening the plants that have compund potentially for cervical cancer therapy. The kit designed with plasmid that contain with HPV 16 or HPV 18 promoter. Each promoter to be accompained by two types of fluorescence reporter such as green (GFP) and red (RFP). The reporter compound obtained from biobrick. GFP obtained from Bba_E0240 (Plate 4, well 11N), then RFP obtained from Bba_K516132 (Plate 1, well 11A). Plasmid also designed with another compund such as RBS and double terminator. GFP act as a marker for the detection viruses whether plasmids have been inserted in hela cells and RFP as a marker for checking the ability of herbal compound used in the treatment of cervical cancer.

Herbs that potentially as a therapy of cervical cancer in the kit are marked by the absence of green or red colors, while the negative results are marked by the emergence of green or red in the kit. The construct in the kit design is herbs can inhibit HPV promoter so that promoters cannot work anymore. The kit is expected to provide convenience to the researchers in the field of cervical cancer and cervical cancer effects can be mitigated with natural compound that not a lot of side effects.

“LAB JOURNAL”

• PREVENTING
A. MAY
• Week 3
Discussion. Discussion about biobrick and what we can do by used this biobrick.
• Week 4
Discussion. Discussion about project - fix about cervical cancer.
B. JUNE
• Week 1
Discussion. Discussion about project - fix that we will do research in “preventing, screening and therapy” for cervical cancer. And our sub-team focus in preventing cervical cancer.
• Week 2
Bioinformatic. Study bioinformatic and protocol for each project.
• Week 3
Bioinformatic. Study bioinformatic for each project and discussion about human practice.
• Week 4
Laboratory. Study in laboratory - learn how to work with gene (competent cell, transformation, etc.)
C. AUGUST
• Week 1
Laboratory. Having traine about working in molecular biology, especially in using lab tools (PCR, Electrophoresis, etc.)
• Week 2
Discussion. Discussion about each project development, human practice, and future plan
• Week 3
Preparation. Competent cell preparation
• Week 4
Preparation. Competent cell preparation
D. September
• Week 1
Discussion & Laboratory. Take hela cells from Yogyakarta and did transformation. Cultured hela cell and discuss with Prof Nellen from Kassel university about funding. Prof Nellen also works at molecular biology for school and the public in Kassel University, it is a student club. He told us how to make an event for funding our project, beside he suggess us to have a good relation and collaboration with other university.
• Week 2
Discussion. Discussion about protocol and wet lab work timeline. We face a problem to choose what is the best promotor for the siRNA. We confused in using CAMP or U6/H1 promotor. Then we discuss it with our beloved supervisor, Mr. Widodo, he give us other promotor named pUR 6 and ask to check it in NetCutter.
• Week 3
Laboratory. Annealing oligonucleotide of siRNA
• Week 4
Laboratory. Transformation to DH5α
E. OCTOBER
• Week 1
Laboratory. Cloning and PCR oligonucleotide.
• Week 2
Laboratory. Electrophoresis.



• THERAPY
A. AUGUST
• Week 3
Lab Preparation. We learn more about protocol that we need. Restriction and Ligation protocol from iGEM protocol and adjust it.
• Week 4
Growing HeLa cells. Thawing HeLa cells from freezing cells. Growing into two flask and incubate in 37ᵒC.
B. SEPTEMBER
• Week 1
Splitting HeLa cells. The confluence HeLa cells (80 – 90%) splitting into another flask then incubate in 37ᵒC.
• Week 2
Cell observation. The splitting HeLa cells observed to control the growth.
• Week 3
Order Synthetic Gene. Order P97 and P105 promoter sequence to IDT (Integrated DNA technology).
• Week 4
Cloning. Insert the P97 and P105 promoter sequence into pJET vector (helped by ITB iGEM Group) then cloned.
C. OCTOBER
• Week 1
Cloning. Restriction the P97 and P105 promoter sequence from pJET vector using EcoRI and Xbal. Beside that, also digest the pSB1C3 backbone using same restriction enzyme. Then, electrophoresis the digested sequence and ligase with pSB1C3 plasmid backbone.
• Week 2
Cloning. Electrophoresis again and then using gel extraction to dissolve the gel. After that, ligase the P97 or P105 promoter sequence into pSB1C3 plasmid backbone.
• Week 3
Submit part to iGEM



• Screening

“Submited Parts”

• Therapy

E0240_P105
We upgraded the standard biobrick backbone pSB1C3 with added by promotor sequences of p105 and GFP (E0240). This pSB1C3 has contained chloramphenicol resistance. Our major achievements is to confirm that herbal compound can react with promotor of HPV 16 and 18 in HeLa cells. GFP give green colour to confirm whether herbal compound can attach and stop the HeLa cell activity transformed with p105 promotor sequence




E0240_P97
Standard biobrick backbone pSB1C3 containing chloramphenicol resistance. The backbone added by promotor sequences of p97 and GFP (E0240). GFP act as a marker that give green colour to confirm whether herbal compound can attach and stop the HeLa cell activity transformed with p97 promotor sequence.

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