Enhanced Lumazine Synthase (ELS) Expression Construct
The part was synthesized by Bio Basic Inc. into the pET28a plasmid vector (which allows for expression of ELS with an N-terminal His-tag). The Enhanced Lumazine Synthase (ELS) was moved into the pSB1C3 vector by the Lethbridge 2011 team.
Lumazine synthase (LS) from Aquifex aeolicus forms icosahedral microcompartment (MC) assemblies of 60 or 180 monomeric units that self assemble and are capable of isolating proteins from their local environment. As shown in previously published work (1), the LS protein has been mutated so that the interior of the MC is negatively charged; the UL 2009 iGEM team has submitted the mutated LS gene to the parts registry (BBa_K249002). A negatively charged interior allows for preferential compartmentalization of positively charged molecules, which can easily be engineered through the addition of a poly-arginine tag to a target protein (1). The size of the cavity of the enhanced Lumazine Synthase microcompartment was enlarged and the negative charge was increased via directed evolution (2). The compartment formed by this polypeptide has a larger cavity and a larger net negative charge, thus allowing a larger loading capacity into the cavity of the compartment.
Part was assembled from intermediate parts.
Double terminator consisting of BBa_B0010 and BBa_B0012
The Enhanced Lumazine Synthase (ELS) sequence was the result of directed evolution. The part was synthesized by Bio Basic Inc. and moved into pSB1C3 by the Lethbridge 2011 team. W??rsd??rfer, B., Woycechowsky, K.J., and Hilvert, D., (2011). Directed Evolution of a Protein Container. ''Science.'' 331: 589-592.
The part was synthesized by Bio Basic Inc. into the pET28a plasmid vector with an N-terminal His-tag. The Enhanced Lumazine Synthase (ELS) without the His-tag was moved into the pSB1C3 plasmid vector by the Lethbridge 2011 team. The size of the cavity of the Lumazine Synthase microcompartment was enlarged via directed evolution (W??rsd??rfer ''et al.,'' 2011). The compartment formed by this polypeptide has a larger cavity than the Lumazine Synthase microcompartment submitted by the Lethbridge 2009 team ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K249002 BBa_K249002]). A larger interior allows for the option of localizing larger and/or more proteins into the cavity of the compartment. Reference: W??rsd??rfer, B., Woycechowsky, K.J., and Hilvert, D., (2011). Directed Evolution of a Protein Container. ''Science.'' 331: 589-592.
The compartment formed by this polypeptide has a larger cavity than the Lumazine Synthase microcompartment submitted by the Lethbridge 2009 team ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K249002 BBa_K249002]).
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.
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.
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1