Team:ITB Indonesia/Project/PartsAndDevice

These are biobricks that team ITB_Indonesia submitted to Part Registry

4.1 Chasis

Our team use E. coli strain DH5α as a chassis for plasmid cloning and E. coli strain BL21DE3 as chassis for aflatoxin whole cell biosensor systems. E.coli cells BL21DE3 chosen because this a strain have RecA genes that become part of a whole cell biosensor systems Aflatoxin.

4.2 Backbone

We use pSB1C3 and pSB1K3 as backbonds in order to suitable witf RFC system, also our team use  pSB1C3 for submite our part to part registry. 3A assembly system was used in process assembling the part, so we use pSB1K3 that have different marker selection with pSB1C3.

4.3 Part/Composite

4.3.1 BBa_K1064004 (CYP3A4 coding sequens)

CYP 3A4 sequence takes from human genom (accession number : NM_017460.5). we analyse this sequence by software www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=iprscan-120130827-135800-0761-879660.pg. We are also change some base nucleotide to make ensure the illegal restriction site CYP.

This sequence consist of signal peptide signal, although that is still vague (software analysis, not perfome yet experimentally). We have to cut the signal peptide  in order to ensure the CYP protein localize in cytoplasm.  The localization of CYP in cytoplasm make the oxidation of entered aflatoxin B1 produce aflatoxin B1 oxide held in cytoplasm efficiently. AFB1 oxide very reactive toward DNA and unstable form. We are also change some base nucleotide to make ensure the illegal restriction site CYP without signal peptide  

4.3.2 BBa_K1064000 (constitutive promoter + strong RBS + cyp3A4 + double terminator)

For expression CYP3A4 gene (BBa_K1064004) we design the syntetic CYP3A4 gene with addition constitutive promotore (BBa_J23119), strong RBS (BBa_B0030), and double terminator (BBa_B1006). This part is expression constituvely because we want this system ready for aaflatoxin detection.

4.3.3 BBa_K1064002 (pSOS + GFP Generator)

For reporting modul, we use GFP generator (BBa_E0840) under the control pSOS promotor (BBa_J22106). Our team assemble both of part becoming a new part (BBa_K1064002). pSOS promotor is a promotor that regulation SOS response, this promotor will active when LexA (a protein repressor in operator) cleavage by RecA protein. When DNA was damage, Rec A protein will active by binding with ssDNA that produce when replication stop in DNA damage site. The active RecA protein will cleavage LexA (a dimer protein) that binding in operator site, this phenomenon made promotor SOS will expression the gene under it regulation

4.3.4 BBa_K1064003 (pUV + GFP Generator)

This composite has a same function with BBa_K1064002. We assemble pUV promotor (BBa_I765001) with GFP generator becoming a new part. Similar with pSOS promotor, pUV promotor will expression gene under it regulation when trigger by DNA damage.

4.3.5 BBa_K1064005 (pSOS + mCherry)

Another system for reporting modul is composite  BBa_K1064005. We use mCheery (BBa_J06702) as reporter This chromogen help this system to observed by naked eye.

4.3.6 BBa_K1064006 (pUV + mCherry)

This part have a same function with BBa_K1064005