Snowdrift 2 , using YcdB. A module to secrete B-galactosidase using the TAT-export Pathway
source- biobricks torA,lacZ, pLac - eschercihia coli GFP- aequoria victoria
This is a module that contains the following components: 1. A constituive promoter 2. RBS (ribosome binding site) 3. An export tag viz. torA 4. Lac Z gene (Gene for enzyme B-galactosidase) 5. A reporter gene viz. GFP 6. TERM this is an eternal co-operator strain...
a suitable export tag was synthesizd and added in frame. the export tag was designed based on the twin arginine translocation (tat) pathway from E.coli
Swetha Srinivasan, Samit Watve, Mandar Phatak, Chinar Patil
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.
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]
lacZ (encoding beta-galactosidase, full-length)
PCR amplified and standardized from BL21
LacZ encodes beta-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. Beta-galactosidase can also cleaves X-gal and ONPG into colorful products.
YcdB - This part is a export tag that utilizes the Twin Arginine Transport pathway(TAT)
The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin Fe(III)-protoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene
We had to add a base at the end of the tag in such a way that when it was fused with the gene of the protein to be secreted, it would be an in-frame after the scar that would result due to standard assembly. we therefore chose to add a 'C' to the end of the designed export tag to allow it to be fused in frame. this caused an addition of alanine when translated. we checked the changes in secondary structure that this addition caused by the available online tools like SOPMA and SSpro. no significant changes were predicted
Conversion of the iGEM parts registry to SBOL2.1