Green Firefly Luciferase and LRE (under pBAD) L. Cruciata (E. coli optimised)
This is the lux operon from V. fischeri expressed under a pBAD promoter. It emits light when induced by arabinose. luxCDE create a substrate which the luciferase complex of luxA and luxB uses to emit light at a blue wavelength.
Removal of existing lux promoter, and replacement with pBAD
From pHK555 provided by James Slock ( http://departments.kings.edu/biology/lux/bacterial.html )
Luciferin Regenerating Enzyme (L. cruciata)
P22 c2 inverter (Q04530) directing transcription of mRFP (I13507). Constructed with standard assembly except PCR from VF and VR primers was used to generate Q04530 fragment with four-part inverter. Cells carrying M30002 are red, however because PCR was used for cloning there may be silent mutations in the insert. M30002 is clone #2. A second clone will be saved as M30001.
σx Generator + PcomFA-E0240
Ng Siu Wang Edward, Jason Jang, Joo Min Jung
SigmaX generator, SigmaX-inducible Phelicase encoding GFP
Both comX gene and Phelicase are found in genomic sequence of Streptococcus pneumoniae NCTC7465 strain, while the RBS+GFP+DTT, downstream of PCelA, is BBa_E0240. The promoter encoding comX is BBa_K880005 and the double terminator for it is BBa_B0015
Orange Firefly Luciferase (under pBAD) L. Cruciata (E. coli optimised)
This is a TetR protein generator regulated by the constitutive promoter J23106. The protein has a LVA tag for degradation.
tetR promoter+rbs+scfv-FecA 527+rbs+FecI+rbs+FecR'+terminator
This part expresses scFv-fused FecA' protein, FecI, and FecR' protein. Fusion is made at aa 527 of FecA' protein. All are control of tetR promoter.
434 cI (LVA-) Generator (pBAD/araC promoter)
Adding arabinose activates expression of 434 cI(LVA-)
RBS+GFP for expression in Bacillus subtilis
It is succesfully joined B.subtilis RBS with GFP reporter protein. Can be used to measure strenght of B.subtilis promoters. In order to work with it you must first add appropriate terminator at the end. We recommend BBa_K780000 - a terminator designed for use with B.subtilis.
RBS was taken from 'host-takover module' from B.subtilis Bacteriophage SPO1 and GPF is derived from jellyfish Aequeora victoria (BBa_E0040).
This part contains a promoter regulated by LacI, an RBS, coding sequences of FecI and FecR, and terminator.
Expression of lambda cI (LVA) is driven by the OmpC promoter. The OmpC promoter is active when the transcription factor OmpR is phosphorylated. If using Cph8 (I15010) as the OmpR regulator, this device is active in the dark. This device functions as a partial repressor device for I15030 and J13030
Composite part for FecA' (promoter::rbs::FecA')
This part contains a promoter regulated by LacI, a RBS, and coding sequence of FecA' (FecA without PstI site)
The Beta-carotene 15'15'-monooxygenase generator from the D. melanogaster ninaB gene, cleaves beta-carotene into two retinal molecules. Used together with BBa_274210 (beta-carotene generator) it should induce retinal biosynthesis in e. coli.
A constitutive sigma-70 promotor was chosen. Terminator is lacking on this part.
[link:https://dgrc.cgb.indiana.edu/product/View?product=4630 Drosophila genomics ressource centre], Indiana University. Found via [link:http://www.flybase.org flybase], ID: [link:http://flybase.org/reports/FBcl0129473.html FBcl0129473]
Lambda cI (LVA) is expressed under control of the lacI repressible promoter R0011.
This is a firefly luciferase gene under the control of a strong constitutive promoter.
strong promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
* promoter and RBS from partsregistry * luciferase from pGL4.10luc2 (Promega)
Red Firefly Luciferase and LRE (under pBAD) L. Cruciata (E. coli optimised)
Lambda cI Generator (PLlac 0-1 promoter )
Expression of Lambda cI (LVA+) can be controlled by IPTG in LacIq strain.
RFPmcherry expression system in Bacillus subtilis
tetR promoter+rbs+scfv-FecA 155+rbs+FecI+rbs+FecR'+terminator
This part expresses scFv-fused FecA' protein, FecI, and FecR' proteins. Fusion is at aa 155 of FecA' protein. All are under control of tetR promoter.
Inducible 434 cI Repressor Generator (Controlled by IPTG and LacI)
This part will produce 434 cI Repressor when IPTG is introduced into the system. The system must also be free of p22 cII for 434 cI repressor to be expressed.
The absence of p22 cII (constitutive promoter) will yield a constant promotion of LacI, which inhibits the expression of lambda cI. Once IPTG is introduced into the system, it will bind to the LacI, resulting in expression/promotion of 434 cI repressor via the QPI (Quad Part Inverter). Hence, a switch controlled by IPTG is created.
PC and AraC from the AraBAD operator. PC and AraC are on the complementary strand, reading right to left.
tetR promoter+rbs+scfv-FecA 567-576+rbs+FecI+rbs+FecR'+terminator
This part expresses scFv-fused FecA' protein, FecI, and FecR' proteins. FecA' aa 567 to 576 are deleted and replaced by scFv. All are under control of tetR promoter.
lambda cI inverter (Q04510) directing transcription of mRFP (I13507). Constructed with standard assembly except PCR from VF and VR primers was used to generate Q04510 fragment with four-part inverter. Cells carrying M30004 are pink, however because PCR was used for cloning there may be silent mutations in the insert. M30004 is clone #2. A second clone will be saved as M30003.
Scarlet Firefly Luciferase (under pBAD) L. Cruciata (E. coli optimised)
We used this construct to test pBAD strong promoter strength by measuring RFP fluorescence.
Yellow Firefly Luciferase (under pBAD) L. Cruciata (E. coli optimised)
tetR promoter+rbs+scfv-FecA 522-532+rbs+FecI+rbs+FecR
This part expresses scFv-fused FecA' protein, FecI, and FecR' proteins. FecA' aa 522 to 532 are deleted and replaced by scFv. All are under control of tetR promoter.
Generator for TetR-4C and lambda cI - Gal4 fusion protein
This is a generator to create both TetR-4C and lambda cI - Gal4 fusion protein. It allows one PoPS-in to control two biological ANDs.
medium strong constitutive promoter + luc
This is a firefly luciferase gene under the control of a medium strong constitutive promoter.
medium strong promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
This part uses promoter R0051, a strong promoter which is constitutive in the absence of the lambda cI repressor protein, to drive expression of the tetracycline repressor from the paskiba3 plasmid.
This BioBrick contains a ''virA'' receptor from ''Agrobacterium tumefaciens'' C58 under the control of a constitutive promotor. We used it to mutate ''virA'' by error-prone PCR. For screening the mutants BBa_K389011 is also needed.
The ''virA'' gene is under the control a medium strong constitutive promotor.
tetR promoter+rbs+scfv-FecA 571+rbs+FecI+rbs+FecR'+terminator
This part expresses scFv-fused FecA' protein, FecI, and FecR' proteins. Fusion is at aa 571 of FecA' protein. All are under control of tetR promoter.
Expresses B-carotene monooxygenase on a constitutive promotor
This generator expresses a beta-carotene 15,15'-monooxygenase,on a constitutive promotor, that catalyses retinal production from beta-carotene via the following reaction: beta-carotene + O2 = 2 retinal
The brick requires B-carotene supply to function. This can either be supplied externally, or alternatively the BBa_K274210 B-carotene generator will induce full B-carotene synthesis in E. coli.
The ninaB gene was originally cloned from drosophila melanogaster, and cDNA was aquired for production of the biobrick from the Drosophila Genome Ressource Center. Promotor+rbs brick BBa_J13002 and terminator BBa_B0015 were used.
Oxygen-tolerant hydrogenase (with RBS and terminator)
Emma Marriott, Jessica Lam, Tatum Banayat
Hydrogenase is an enzyme that catalyzes the reversible oxidation of H2. This part encodes a hydrogenase actuator taken from the large subunit of the marine bacterium Hydrogenovibrius Marinus, which has been shown to be relatively tolerant to oxygen (Kim et. Al, 2011). This part consists of a strong bicistronic design RBS (found under BioFAB ID: pFAB1655), a codon-optimized HmMBH actuator gene taken from the sequence of the large subunit of hydrogenase found in hydrogenovibrius marinus (original sequence found under F2Z6J6 at uniprot.org), and a high efficiency transcriptional terminator (found under BBa_M36281 in the Registry of Standard Biological Parts). The mechanism of the part is relatively straightforward: Once induced with the PoPS signal, the actuator will initiate protein translation and express the hydrogenase enzyme. Our actuator was used in conjunction with a rhamnose sensor (e.g. BBa_M36284).
Considerations of the benefits and convenience of having a bicsistronic design led us to choose this option, in spite of the fact that the bicistronic design is targeted for E. Coli and has not yet been tested in S. Elongatus. Interestingly enough, although the part was originally designed for expression of the hydrogenase protein in cyanobacteria, it has so far only been proved for the expression of hydrogenase protein in E. Coli. In regards to the HmMBH actuator gene, the sequence was optimized to reduce repeats and the number of sites available to restriction enzymes.
Bicistronic RBS: Mutalik VK, Guimaraes JC, Cambray G, Mai QA, Christoffersen MJ, Martin L, Yua A, Lam C, Rodriguez C, Bennett G, et al. Quantitative estimation of activity and quality for collections of functional genetic elements. Nat. Methods. 2013;10:347-353. BioFAB. (http://io.biofab.org:5000/simple/randomized_bds) Sequence for large subunit Hydrogenase: Nishihara H, Miyata Y, Miyashita Y, Bernhard M, Pohlmann A, et al. Analysis of the molecular species of hydrogenase in the cells of an obligately chemolithoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus. Biosci Biotechnol Biochem. 2001;65:2780???2784. UnitPRO (http://www.uniprot.org/uniprot/F2Z6J6). High efficiency transcriptional terminator: Sunil Bodapati, Julia Joung, Daniel Esquivel-Reynoso. Part: BBa_M36281: Hard Information. Registry of Standard Biological Parts. (http://partsregistry.org/cgi/partsdb/part_info.cgi?part_name=BBa%20M36281)
This is a firefly luciferase gene under the control of a weak constitutive promoter.
weak promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
This is a construct for testing promoter strength by measuring RFP fluorescence.
Inducible Lambda cI Repressor Generator (Controlled by ATC and TetR)
This part will produce Lambda cI Repressor when ATC is introduced into the system. The system must also be free of p22 cII for Lambda cI repressor to be expressed.
The absence of p22 cII (constitutive promoter) will yield a constant promotion of TetR, which inhibits the expression of lambda cI. Once ATC is introduced into the system, it will bind to the TetR, resulting in expression/promotion of lambda cI repressor via the QPI (Quad Part Inverter). Hence, a switch controlled by ATC is created.
tetR promoter+rbs+scfv-FecA 176+rbs+FecI+rbs+FecR'+terminator
This part expresses scFv-fused FecA' protein, FecI, and FecR' proteins. Fusion is at aa 176 of FecA' protein. All are under control of tetR promoter.
A CFP protein generator under control of c1 for use as a negative control with the Synchronator constructs.
This is a TetR protein generator regulated by the constitutive promoter J23111. The protein has a LVA tag for degradation.
Green Firefly Luciferase and LRE L. Cruciata (E. coli optimised)
The part accepts POPS to produce TetR. This sequence encodes the ribosome binding site and tetracycline repressor protein from the plasmid pASKIBA3.
A CFP protein generator under control of Lac for use as a negative control with the Synchronator constructs.
lambda cI inverter (Q04510) directing transcription of mRFP (I13507). Constructed with standard assembly except PCR from VF and VR primers was used to generate Q04510 fragment with four-part inverter. Cells carrying M30003 are pink, however because PCR was used for cloning there may be silent mutations in the insert. M30003 is clone #1. A second clone will be saved as M30004.
P22 c2 inverter (Q04530) directing transcription of mRFP (I13507). Constructed with standard assembly except PCR from VF and VR primers was used to generate Q04530 fragment with four-part inverter. Cells carrying M30001 are red, however because PCR was used for cloning there may be silent mutations in the insert. M30001 is clone #1. A second clone will be saved as M30002.
This part is a differs from BBa_P1011 in that the mutations introduced in BBa_P1011 to remove restriction sites are not present in this part. It does have the "normal" ccd promoter and ccdB coding region. It also has a double TAA stop codon as per BioBrick conventions.
ccdA- was eliminated since it is an inactive form of ccdA and should not be required for the toxic phenotype of ccdB.
This part is derived from BBa_P1010 but it lacks ccdA-.
σx Generator + PcelA-E0240
Jordy Evan, Nadia Benedicta, Angeline Widjaja
SigmaX generator, SigmaX inducible PCelA encoding GFP
Both comX gene and PCelA are found in genomic sequence of Streptococcus pneumoniae NCTC7465 strain, while the RBS+GFP+DTT which is downstream of PCelA is BBa_E0240. The promoter encoding comX is BBa_K880005 and the double terminator for it is BBa_B0015
A CFP protein generator under control of LuxR for use as a negative control with the Synchronator constructs.
This is a construct for testing promoter strength by measuring GFP fluorescence.
This is a TetR protein generator regulated by the constitutive promoter J23109. The protein has a LVA tag for degradation.
The ccd operon having a constitutive promoter and the ccdB coding region. ccdB protein is lethal in normal cloning strains. This part is used to aid the process of moving a BioBrick part to a new plasmid. A target plasmid carrying P1010 is cut and mixed with the cut insert. Plasmids that recombine are selected against because they kill the cell that picks them up. P1010 is only provided as plasmid or in DB3.1 which is ccdB resistant.
This part generates ~98.5% monocistronic CFP and YFP + ~1.5% polycistronic CFP/YFP based on the termination efficiency of B0015.
Generator for wild-type TetR and lambda cI - Gal4 fusion protein
This is a generator to create both wild-type TetR and lambda cI - Gal4 fusion protein. It allows one PoPS-in to control two biological ANDs.
Addition of arabinose activates expression of 434 cI (LVA+)