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Team:UB Indonesia/screening
From 2014.igem.org
Screening
Introduction
Cervical cancer is one of the most common cancers in women worldwide, with an estimated global incidence of 470,000 new cases 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.
Background
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 the protein contains tandem repeats of 34 amino acids sequences (termed monomers) that are required 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)
Next
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:
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
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)
Prototype
