E. coli dxs-GFP protein fusion (lac promoter with LVA-tagged lac inhibitor (LacI:LVA) - IPTG inducib
With the exception of the artificial terminator and the GFP part from BBa_K1088007 (GFP = BBa_E0040), everything derives from E. coli K-12 MG1655
This is the same part as BBa_K1088007, with the exception of LacI under the control of a constitutive promoter to ensure regulation of the Lac Promoter This part consists of 5 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_K1088007
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_K1088007). In the BBa_K1088007 part there is specific sites for scars aswell. Go to that parts page to investigate further.
E. coli dxs-GFP fusion (lac promoter without lac inhibitor: IPTG uninducible)
The subparts of this composite part is derived from E. coli K-12 MG1655. The GFP derives from Aequorea victoria.
This composite part consists of ? subparts: A Lac promoter: BBa_R0010 A strong RBS: BBa_J15001 The Dxs protein from E. coli: BBa_K118000 A 10 aa protein linker: BBa_K105012 A GFP reporter protein: BBa_E0040
We used USER cloning to assemble this part and therefore there are no scar sites. However we did insert a scarsite between the RBS and the start codon aswell as between the promoter and RBS, for optimal expression of our protein.
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.
dxs coding sequence encoding 1-deoxyxylulose-5-phosphate synthase
This is the coding sequence of dxs from Escherichia coli JM109. It encodes 1-deoxyxylulose-5-phosphate synthase, thiamine-requiring, which catalyses teh first step in the biosynthesis of terpenoids. Overexpression of dxs has been reported to increase yields of carotenoids and other terpenoids [add reference here].
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).
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