B. subtilis dxs-GFP protein fusion (lac promoter with LVA-tagged lac inhibitor (LacI:LVA) - IPTG ind
With the exception of the artificial terminator and the GFP part from BBa_K1088008 (GFP = BBa_E0040), everything derives from E. coli K-12 MG1655
This is the same part as BBa_K1088008, with the exception of LacI under the control of a constitutive promoter to ensure regulation of the Lac Promoter This part consists of 7 subparts: A constitutive promoter: BBa_J23106 A strong RBS: BBa_B0030 The LacI repressor protein, regulating the lac promoter: BBa_C0012 An artificial terminator: BBa_B1002 and the rest is similar to BBa_K1088008
The terminator aswell as the RBS and promoter was part of the reverse primer and forward primer for the LacI part, respectively. However we did insert a scar sequence between the RBS and startcodon to improve efficiency. There is also scar sites between the terminator (BBa_10002) and the rest of this composite part (BBa_K1088008). In the BBa_K1088008 part there is specific sites for scars aswell. Go to that parts page to investigate further.
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:LVA device (constitutive promoter, RBS and terminator)
The source of the part will be its components.
The trancsriptional repressor lacI with LVA tag for faster degradation (BBa_C0012)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.
represillator of Elowitz and Leibler (2000)
See BBa_C0012 Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator BBa_R0010 and PLlac01 hybrid regulator BBa_R0011 and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degradation tail (LVA) has been added to improve the switch time for High to Low performance of this part.
Sequence taken from the repressilator of Elowitz and Leibler (2000). The obtained sequence was compared to the wild-type sequence for LacI obtained through a database search. The sequence had been modified from the wild-type in that wild-type GTG start was changed to an ATG start (note, actual ORF in E.coli has several GTG starts it would seem). The LVA tag has been added for quicker degradation. Incompatible with systems containing LacI, lactose, or IPTG.
Conversion of the iGEM parts registry to SBOL2.1