After creating two intermediate parts, the upstream one being the Cadmium Promoter with Ogr Activator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1042012 BBa_K1042012]) and the downstream one being the PO Promoter with GFP ([http://parts.igem.org/Part:BBa_K1042013 BBa_K1042013]), we digested the intermediates and ligated them to the linearized pSB1C3 plasmid provided. The resulting colonies were tested for responsive GFP production from the addition of cadmium.
plasmid DNA supplied by Prof. Richard Calendar, University of California.
The ogr activator taken from P2 phage acts on a class of inducible promoters (parts I746360 to I746365), inducing their activity to varying degrees. The part sequence does already contain a ribosome binding site (B0034)!
The part does contain a RBS (B0034) already.
GFP reporter for RHS of library test constructs
This part will be suffixed to a promoter.RBS library. The purpose of the experiment is to make a first pass at a library-based construction step to identify efficiency issues and also to examine maxPOPS/RIPS level.
This part was built by Jen as an intermediate to their nuts and bolts parts.
CadA promoter which has the binding site for CzrA protein is combined with ArsR binding site to create this AND gate Cadmium sensor.
This Cadmium sensor has binding sites for ArsR and CzrA metal sensors. By placing them together, our device works as an AND gate and senses Cadmium when both proteins bind to Cadmium. When Cadmium is not present both proteins bind to their relevant sites on the promoter and represses the expression of downstream genes. When Cadmium present in the environment, it binds to these repressors which then fall of from where they are bound to on the promoter. Hence the promoter becomes free to express the downstream genes as an indication of Cadmium. This combinatorial approach enables us to filter out sensing other metals. ArsR and CzrA can bind to different metals, however when both are used as an ANd gate, they will only recognize Cadmium.
We wanted to sense Cadmium accurately and used a combinatorial approach to achieve our goal.
The source of the DNA is the P2 phage genome.
This is the PO promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PO promoter.
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.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
This part purely holds the binding site for ArsR protein which can be released when bound to different metals. Hence this part can be combined with any promoter to sense metals that can bind to ArsR protein. When added in front of a promoter with another metal sensor's protein's binding site, it can form an AND gate to sensitively detect specific metals.
It was developed mainly to use combinatorial approaches for metal sensing.
It is taken from ''B. subtilis'' ''cadA'' promoter.
In ''B. subtilis'' CadA system works as an efflux system to take the metals out in the cell. It is normally repressed by CzrA repressor protein. When certain metals get inside the cell, they can bind to CzrA and derepress the promoter expressing CadA. Hence metals are taken out by the efflux system. By taking the promoter of CadA system, we created a generic metal sensor. Hence when it is combined with coding sequences of other genes, it will regulate the expression of these genes upon sensing metals.
It was developed mainly to use combinatorial approaches for metal sensing
Conversion of the iGEM parts registry to SBOL2.1