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Team:ITB Indonesia/Parts
From 2014.igem.org
PARTS
PARTS DESCRIPTION
- Reporter Module
- Degradation Module
- Self-Regulatory Module
- Convertion Module
- References
The construction of reporter module is consist of inducible promoter PibpAB (BBa_K339010), RBS (BBa_B0034), amilCP (BBa_K592025) , and double terminator (Bba_B0015) . BBa_K1387000 consist of RBS (Bba_B0034), amilCP (BBa_K592025) and double terminator (BBa_B0015), while the complete module was contained in (Bba_K1387001). The reporter module will have a blue chromoprotein when the inclusion body is formed in the cell. This mechanism then acts as an indicator of cytoplasmic stress, in this situation is caused by inclussion body.
The casette of degradation module (BBa_K1387006) is comprised of constitutive promoter, tet operator, RBS, outer membrane protein A (ompA) fused with LC Cutinase, and double terminator. By using constitutive promoter, we expect the recombinant protein, in this case ompA-LC Cutinase, will be synthesized indefinitely. OmpA is outer membrane protein, it comprises of β–strand B3-B7. Rather than using full sequence of ompA, we use ompA (46-159) fused with lipoprotein signal sequence, thus exposing it on the cell surface of bacteria1. We fuse lpp-ompA with LC Cutinase, an enzyme that exhibit esterase activity. LC-Cutinase esterase activity will cleave the ester bond on PET and releasing terephthalic acid and ethylene glycol as a product. Fusion strategy of lpp-ompA on LC Cutinase gene is one of our way to create a whole cell biocatalyst which will be an effective molecular machinery to degrade PET on the environment. Due to constitutive expression of the recombinant protein in E.coli, there is a possibility of formation of insoluble protein inside the cell (inclusion body), because of that, we put tet operator upstream of RBS for further regulation (Georgiou et al, 1996).
pIBPAB is a hybrid promoter that induced by inclusion body from unfunctional protein. This promoter followed by strong RBS (BBa_B0034), make a new part called Bba_K1387005
This casette (BBa_K1387002) consist of TetR (BBa_C0040) and Double Terminator (Bba_B0015) . TetR is tetracycline repressor that bind to operator region and repressing the expression of downstream gene. In our system, the degradation module consist of TetO (Tet Operator) which is region of TetR (Tet Repressor) placed.
The construction of reporter module is consist of inducible promoter PibpAB (BBa_K339010)RBS (BBa_B0034), amilCP (BBa_K592025) , and double terminator (Bba_B0015) . BBa_K1387000 consist of RBS (BBa_B0034), amilCP (BBa_K592025) and double terminator (Bba_B0015) , while the complete module was contained in BBa_K1387001 . The reporter module will have a blue chromoprotein when the inclusion body is formed in the cell. This mechanism then acts as an indicator of cytoplasmic stress, in this situation is caused by inclussion body.
Improvisation the part submitted by UC Davis team BBa_K936024 by combining the construct with terminator, so we can characterize the expression of the enzymes and performe the assay for ethylene glycol utilization better than before. We name this BioBrick as convertion module.
1Georgiou,G., Stephens,L., Stathopoulos,C., Poetschke,L., Mendenhall,J., and Earhart,F. 1996. Display of β-Lactamase on Eschericia coli Surface : Outer Membrane Phenotypes Conferred by Lpp’-ompA’- β-Lactamase Fusions. Protein Engineering, 9, 239-247.
