Plac+B0034+alsS+zif268+B0034+PBS II+ilvC+B0034+HIVC+ilvD+Ptet+B0032+kivD+B0015
The circuit is optimized from 2011 NCTU_FORMOSA.
This part contain four enzymes. Each of the former three enzymes(alsS, ilvC& ilvD) linked with a zinc finger. The three enzymes could convert pyruvate into 2-ketoisoalerate(the Intermediate of isobutanol). The final enzyme(kivD)could change 2-ketoisoalerate into isobutyraldehyde, which would be converted into isobutanol by ADH in E.coli. This is a complete circuit to produce isobutanol.
We add zinc fingers in front of enzymes in the circuit, and therefore the enzymes could bind the DNA program in order. It help enhance the efficiency of isobutanol production .
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Transcriptional terminator consisting of a 64 bp stem-loop.
AlsS (alpha-acetolactate synthase protein)
This is the coding region of AlsS from Bacillus subtilis. It encodes alpha-acetolactate synthase protein which is part of isobutanol biosynthesis pathway.
Because of the four RE site, EcoR1, Xba1, Spe1 and Pst1, before and after the coding sequence, those RE site should not exist in coding sequence. There are two Pst1 site, one Xba1 site and one EcoR1 site in original sequence of AlsS. However, those RE site in AlsS has been modified by point mutation.
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Double terminator consisting of BBa_B0010 and BBa_B0012
kivd (alpha-ketoisovalerate decarboxylase)
It is cloned form Lactococcus lactis subsp. lactis KF147
kivd is the coding sequence from Lactococcus lactis subsp. It encodes alpha-ketoisovalerate decarboxylase which is responsible for the decarboxylation into aldehydes of α-keto acids derived from amino acid transamination. This is part of the isobutanol biosynthesis pathway.
In order to design the four RE site, EcoR1, Xba1, Spe1 and Pst1, before and after the coding region.However, there is one RE site, EcoR1, in kivd. The RE site in kivd has been modified by point mutation.
ilvC (ketol-acid reductoisomerase NAD(P)-binding)
It comes from Escherichia coli str. K-12 substr. MG1655
This is the coding region of ilvC from Escherichia coli str. K-12 substr(MG1655). It encodes ketol-acid reductoisomerase NAD(P)-binding which is part of the isobutanol biosynthesis pathway.
Because of the four RE site, EcoR1, Xba1, Spe1 and Pst1, before and after the coding gene, we have to delete those RE site in kivd. The RE site in the part has been modified by point mutation.
This is the coding sequence of ilvD form Escherichia coli str. K-12 substr. MG1655. It encodes dihydroxyacid dehydratase which is part of isobutanol biosynthesis pathway.
Because of the four RE site, EcoR1, Xba1, Spe1 and Pst1, before and after the coding sequence, those RE site should not exist in coding sequence. There is one Pst1 in original sequence of ilvD. However, the RE site in ilvD has been modified by point mutation.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Conversion of the iGEM parts registry to SBOL2.1