Dxs from B. subtilis with lac promoter and lacI (IPTG inducible)
The part consist of the dxs gene derived from B. subtilis under the control of the lac promoter and has a strong RBS. To repress expression from the lac promoter, the lacI gene under a constitutive promoter, with a strong RBS and a efficient terminator is placed counter-clockwise to the reporter fusion. The repression can then be relieved with addition of IPTG, which binds and inhibits the function of LacI. The purpose of the brick is to increase the flow through the MEP pathway upon addition of IPTG.
The lac promoter was used to enable control of the expression, since overexpression of the dxs gene has proved to lower the growth rate.
LacI device (constitutive promoter, RBS and terminator)
The source of the part will be its components.
The trancsriptional repressor lacI (BBa_K1088018)under the constitutivly active promoter (BBa_J23106), with a RBS (BBa_B0030) and with a terminator (BBa_B1002). This device can be used to overexpress lacI with the purpose of repressing transcription from high-copy plasmids containting the lac promoter (BBa_R0010). The repression can be relieved by allosteric binding of IPTG to lacI, thereby promoting transcription from the lac promoter.
Used a strong terminator so that the composite part can be used in front of the lac promoter without unwanted transcription of the regulated gene.
B. subtilis dxs (lac promoter without lac inhibitor)
The Lac promoter derives from E. coli. The Dxs protein is from B. sub
This part has the B. sub dxs (BBa_K1088000) controlled by the common Lac promoter. Notice that there is no LacI in this system, which is required for regulation of the Lac promoter.
We forgot to acknowledge the need for high expression of LacI to keep the regulation of the lac promoter vaild. The constructs where assembled with USER cloning, and therefore there are no scarsites between the parts. There are however inserted scarsites between the RBS and the coding region aswell as between the lac promoter and the RBS to improve translational efficiency
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). 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 Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
strong synthetic E. coli ribosome binding site with SacI site.
This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.
Note the presence of a SacI site overlapping the XbaI site, which is preserved when this biobrick is added to any other biobrick. At the time of writing, this biobrick is added as a short piece of DNA composed of two complementary oligonucleotides rather than being incorporated into a biobrick cloning vector by itself. It can be added upstream of a biobrick coding sequence, and its presence can easily be detected in miniprep DNA on a gel by using a SacI-SpeI or similar digest.
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 6 residues.
1-deoxyxylulose-5-phosphate synthase (Dxs) enzyme from MEP pathway isolated from B. subtilis 168
The MEP pathway is a common bacterial isoprenoid pathway. To improve the yield of the end product, isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate(DMAPP), Zhao et al. expressed, in 2011, the 1-deoxyxylulose-5-phosphate synthase(Dxs)gene aswell as the second step of the MEP pathway. They saw a 2.3 fold increase in isoprene output (isoprene is one of many products from the MEP pathway). From Biocyc: "Dxs catalyzes the thiamin diphosphate-dependent reaction that condenses pyruvate and D-glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose 5-phosphate [Julsing07]. This is the first, rate-limiting step in the methylerythritol phosphate pathway of isoprenoid biosynthesis, and is believed to feed into the pyridoxal 5-phosphate (vitamin B6) and thiamin (vitamin B1) biosynthesis pathways." -http://biocyc.org/BSUB/NEW-IMAGE?type=ENZYME&object=BSU24270-MONOMER
we changed TTG start codon to ATG to improve translation efficiency. We performed 4 silent site directed mutageneses to remove 2 EcoR1 and 2 Pst1 restriction sites
Mixed site (ACTAGA)when XbaI and SpeI sticky ends ligate
>gnl|ECOLI|EG10525 lacI "PD00763" (complement(366734..365652)) Escherichia coli K-12 substr. MG1655 http://ecocyc.org/ECOLI/sequence-rc?type=GENE&object=EG10525
Coding region for the LacI protein. LacI binds to the lac promoter BBa_R0010 and inhibits transcription. IPTG binds LacI and inhibits its function, and thus promotes transcription from the lac promoter.
Conversion of the iGEM parts registry to SBOL2.1