EfeU iron (II) transporter + 5' UTR (medium) + terminator + Fur promoter + Gemini
This composite part has our actuator and sensor combined into a single sequence, followed by Gemini.
EfeU elemental iron (II) transporter + 5' UTR (medium) + terminator + Fur promoter
This composite part has our actuator and sensor combined into a single sequence.
After the terminator of the actual part, we have the Fur promoter. We're planning to place a universal sensor before this part (to trigger expression of EfeU) and a universal actuator after (to trigger expression of a signal when iron is low).
Gemini, a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression. PLOS One. Lance Martin et al.
A medium strength RBS expressing Gemini, ending with a strong terminator.
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Transcriptional terminator consisting of a 64 bp stem-loop.
1. Gunter K, Toupet C, Schupp T. Characterization of an iron-regulated promoter involved in desferrioxamine B synthesis in Streptomyces pilosus: repressor-binding site and homology to the diphtheria toxin gene promoter. Journal of bacteriology. 1993;175(11):3295-302. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=204726&tool=pmcentrez&rendertype=abstract.
pPS-PCR is a promoter which is controlled by the iron-repressive transcription factor, Fur.
Based on deletion analysis of natural S. lividans gene
EfeU elemental iron (II) transporter + 5' UTR (medium) + terminator
This composite part has the EfeU iron transporter downstream from a medium 5' Bicistronic UTR. There is a terminator at the end.
We are hoping that this 5' UTR will cause enough expression of the EfeU protein to measure, without producing so much that the organism dies due to excessive iron uptake or other ion imbalances.
amino acids 3-60 from lacZ and amino acids 2-end of GFP (E0043)
This part is a fusion protein between GFP and the alpha fragment of lacZ. It can be measured via fluorescence from GFP or through an assay for beta-galactosidase in a strain containing the omega fragment of lacZ.
The amino acid linker AGGSEGGGSEHHHHHHGSE is between the lacZa and GFP. The 6-his can also facilitate detection on a Western blot, for purification, etc.
5' Bicistronic UTR (medium), does not include ATG start
Vivek Mutalik et al. c/o BIOFAB Emeryville.
5' UTR based on bicistronic junction architecture. First RBS is strong and drives expression of short leader cistron. Second RBS internal to first cistron control translation of gene of interest. Architecture is optimized so that downstream CDS for gene of interest does not have a chance to form a secondary structure with the 5' UTR. See BIOFAB.org for more information.
Guillaume Cambray et al. c/o BIOFAB Emeryville
E. coli RNA pol transcription terminator designed and tested by Guillaume Cambray at BIOFAB Emeryville. Based on the natural E. coli rnpB T1 terminator.
From wild-type Nissle 1917 E. coli. The sequence came from the Ecogene entry EG13860.
This is the reconstructed gene for the EfeU iron transporter. This gene is inactive in K-12 strain E. coli, but has been characterized in the Nissle 1917 strain. (Grobe, Scherer et al. PMID: 16987175.)
The sequence was conceptually reconstructed from the frame-shifted version. The Ecogene entry has an "N" for the nucleotide which is uncertain, but a comment in that entry says that the correct amino acid is likely glycine. Thus we chose N = G in the final sequence.
Conversion of the iGEM parts registry to SBOL2.1