Team:UB Indonesia/backup
From 2014.igem.org
According to Bosch FX (2003), 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.
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. :
Background
Detail.
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.