LacI Promoter Variant #5 in Characterization Construct
Mutagenic PCR of the LacI repressible promoter, R0010, and standard BioBrick assembly of parts from the registry.
This part contains a variant of the wild type LacI repressible promoter, and is followed by the LacI characterization construct, K611013. This allows for testing of this particular mutant promoter at various concentrations of repressor. The mutated LacI promoter in this plasmid expresses GFP and is followed by an arabinose inducible promoter, pBad, which expresses the LacI repressor. Thus, a user can increase the concentration of repressor by inducing pBad with arabinose. Similarly, the repressor may be unrepressed with the addition of IPTG. This plasmid is very useful for the further characterization of K611025.
This part does not have a termination sequence at the end. It is recommended that for optimal results, this part be used in a backbone that includes a terminator (such as pSB1C3).
Built from existing parts in the registry though BioBrick assembly.
This part is used to characterize mutants of BBa_R0010 (pLacI). By adding a mutant of BBa_R0010 before this part, one can measure several of its properties through the expression of GFP. Certain characteristics of the mutant, such as the binding of the LacI repressor, can be measured after inducing the pBad promoter with arabinose. Concentration of the repressor can be varied through different arabinose concentrations as long as AraC is present.
There is no terminator at the end, so the part needs to be used in a plasmid that has a terminator after the BioBrick suffix (such as pSB1A3 or pSB1C3). This part contains the PBad promoter without AraC, thus, a strain that already includes AraC is recommended in order to induce PBad.
Parts K611021 through K611027 are variants of the wild type LacI repressible promoter, BBa_R0010. These parts were generated through three rounds of mutagenic PCR and each have around 1 to 7 mutations. Each of these variants differ in properties such as transcriptional strength and repressor binding affinity and have been well characterized. This can be very useful in tuning genetic circuits for specific outputs.
We chose to mutate this part from a well known promoter so that any changes in behavior would be easy to observe.
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.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Double terminator consisting of BBa_B0010 and BBa_B0012
represillator of Elowitz and Leibler (2000)
Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator <bb_part>BBa_R0010</bb_part> and PLlac01 hybrid regulator <bb_part>BBa_R0011</bb_part> and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degredation tail (LVA) has been added to improve the High to Low performance of this part.
References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] 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.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1