B. subtilis dxs-GFP protein fusion (lac promoter with lac inhibitor: IPTG inducible)
This part consist of the dxs gene derived from B. subtilis fused to GFP at translational level with a 10 AA linker between the proteins. The reporter fusion is under the control of the lac promoter and has a strong RBS. To repress expression from the lac promoter, the lacI under a constitutive promoter, with a strong RBS and a efficient terminator is placed counter-clockwise to the reporter fusion. The repression can be relieved with addition of IPTG, which binds and inhibits the function of LacI. The levels of expression was meassured in E. coli K-12 MG1655 using fluorescence activated cell sorting, and proved that their was no expression when grown without IPTG and that there was expression after addition of IPTG. See experience for more details. This Brick was build to test induction time and IPTG concentration for induction of a similar device which lacks the linker and GFP (BBa_K1088027)
A linker was placed between the two genes to decrease the risk of misfolding and loss of function.
B. subtilis dxs-GFP fusion (lac promoter without lac inhibitor: IPTG uninducible)
The parts mainly derive from E. coli K-12 MG1655 but the GFP as an exception comes from Aequorea victoria
This composite part consists of 5 parts A lac promoter: BBa_R0010 A strong RBS: BBa_J15001 The Dxs from B. subtilis: BBa_K1088000 A 10 aa protein linker: BBa_K105012 A GFP reporter protein: BBa_E0040
The system was assembled with USER cloning but we inserted a scar site between the RBS and start codon aswell as between the promoter and the RBS. We did this to improbe translational efficiency. We used the terminator in the pSB1C3 plasmid.
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.
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.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
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.
Oligos with a yeast optimized coding for the peptide GENLYFQSGG have been ordered <br\n> The amino acide sequence corresponds to the interdomaine linker of xxx. ''paper''
This is a 10 amino acides long linker peptide which can be used to join protein domains together in a flexible way. So fusion proteins with variable DNA-binding and activation or repression domains might be assembled. <br\n>
To enable the fusion with other domains in frame the vector of this BioBrick has no base pair in between the restriction side and the BioBrick. Furthermore, this coding sequence does not include a start codon.<br\n> For more information about this issus, see:<br\n> Phillips, I.E. and Silver, P.A. "A new Biobrick Assembly Strategy Designed for Facile Protein Engineering." <br\n> DSpace http://hdl.handle.net/1721.1/32535 (2006).
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
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 6 residues.
>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