The majority of the subparts come from existing parts in the registry. We also designed a lead uptake part (Bba_J92000) which transports lead into the cell.
Our device has two main functions. First of all, it acts as an environmental lead sensor, alerting the user to the presence of lead 2+ ions. Secondly it bioaccumulates the lead ions and removes them from the solution.
In order to design this part we used the standard 23. We used the NEB biobrick assembly kit for this standard.
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.
This 5.7Kbp part contains 11 open reading frames coding for gas vesicle genes (gvpB,R,N,F,G,L,S,K,J,T and U) from Bacillus megaterium VT1660 AF053765. Original AF053765 DNA in pBluescriptIIKS plasmid (pNL29 in "Gas Vesicle genes identified in Bacillus megaterium and Functional Expression in Escherichia coli, Ning Li and Maura C. Cannon Jounal of bacteriology May 1998 p2450-2458).
This 5.7Kbp part contains 11 open reading frames coding for gas vesicle genes (gvpB,R,N,F,G,L,S,K,J,T and U) from Bacillus megaterium VT1660 AF053765. Promotion of the sequence results in expression of gas vesicles which slow the rate of settling of the cells in Saline.
Site directed mutagenesis was performed in four rounds to remove 3 PstI sites and one EcoRI site from gvpL in the sequence. Primers with biobrick prefix and suffix were then used in a PCR reaction to amplify the part. Finally BBa_J61035 was used to provide the plasmid backbone which introduced a RBS at the front of the part.
10 repeats of 40bp induced durring mutagenesis
Overlap extension of synthetic oligonucleotides
Lead Promoter (Part 4) messed up EcoRI site
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs
Artificial terminator, estimated %T~>90% *8bp stem, 6nt loop *Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 9 residues.
Borremans,B., Hobman,J.L., Provoost,A., Brown,N.L. and van Der Lelie,D. "Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34" J. Bacteriol. 183 (19), 5651-5658 (2001)
This part was derived from the Pbr T lead uptake protein from Ralstonia metallidurans. The protein is responsible for the uptake of Pb 2+ ions. This part can be used for projects where one needs to uptake lead ions from the environment into the cell interior.
When designing the part we checked the gene sequence/restriction site profile to make sure the part would be compatible with standards 10 and 23.
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. This Composite Biobrick is created by standard assembly of Part BBa_K118014, Part BBa_K118006 and Part BBa_K118005.
This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs) and Part BBa_K118005 (CrtI with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance). Together, enzymes CrtE, CrtB and CrtI convert colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). The red lycopene pigment can then be used as a coloured signal output, e.g. for biosensors. There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006 and BBa_K118005 for more information on individual Biobricks components. This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274110) and arabinose-inducible promoter I0500 (see Part BBa_274120), transformed and tested in E.coli strain MG1655. Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that the Composite Biobrick, when put under a promoter, works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (lycopene) production than strain TOP10.
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.
crtE (geranylgeranyl pyrophosphate synthase) coding sequence.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
crtE (geranylgeranyl pyrophosphate synthase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis parthway.
crtB coding sequence encoding phytoene synthase
This is the coding sequence of crtB from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene synthase, part of the carotenoid biosynthesis pathway, which converts geranylgeranyl diphosphate to phytoene. Reference: Misawa,N., Nakagawa,M., Kobayashi,K., Yamano,S., Izawa,Y., Nakamura,K. and Harashima,K. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol. 172 (12), 6704-6712 (1990)
crtI coding sequence encoding phytoene dehydrogenase
This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene.
The native crtI sequence contains two internal PstI sites. Primers crtImutf1 and crtImutf2 were ordered so that the MABEL protocol (invented for this purpose) could be used to remove these sites. The mutagenic primers were as follows: * crtImutf1: gtt tac agt aag gtt gcc * crtImutf2: agt aac tct ctg ttt gtg c In both cases, the PstI site CTGCAG was converted to CTGCAA, a silent mutation.
Synthetic ribosome binding site added by Son of Babel procedure
This is a 4bp synthetic ribosome binding site which can be added by fusion PCR using a reverse primer and the Son of Babel cloning system.
The part can be added by fusion PCR using a Son of Babel reverse primer.
Conversion of the iGEM parts registry to SBOL2.1