Double terminator consisting of BBa_B0010 and BBa_B0012
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - CpxR-IFP[1] + CpxR-IFP[2] (BBa_K1286008) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
All orientations were made, so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used for each of the fused proteins, and an RBS was put before each one of them.
double terminator (B0012-B0011), reversed
Device expressing the Gb3 Synthase enzyme under control of an arabinose inducible promoter
This part will code for Gb3 synthase. Gb3 synthase in return makes Gb3 which is a receptor for Shiga-like toxin (stx 1&2). Shiga-like toxins are exotoxins, which consist of a toxic enzymatic A subunit and a cell-binding B subunit. The latter binds to a globotriaosylceramide (Gb3) receptor, expressed on the surface of the target cells in humans. This interaction is responsible for the toxin's entry into the host cell. When the receptor is exposed to the food sample, the SLT subunit B, if present, will bind to Gb3.
An inducible promoter (BBa_10500), RBS (BBa_B0034) Scar was added to the sequence. Along with a terminator.
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the C terminus of CpxR).
We had to test both attachments, N and C terminus of the CpxR.
The arabinose promoter and the sfGFP were obtained from the registry, while the CpxR was obtained from the E coli genome.
Two terminators in Bgl operon that is antiterminated by bglG
two start codons coding for RFP and sfGFP reporters
This part was used in a design that tests multiple start codons. The first start codon codes for RFP. The second start codon codes for sfGFP. They are 7 nucleotides apart on the DNA strand. The ribosome binding site translation initiation rate determines which protein is expressed. The part has two restriction enzyme sites flanking the RBS and promoter regions, so varying RBS strengths may be tested.
This design used gBlocks synthesized by IDT. Several CBAR reactions were necessary to achieve the construct.
This part contains no direct BioBrick parts. It was assembled from several pieces of plasmid DNA in the Salis Lab at Penn State. The dRBS1 vector and pBAC reporter components were assembled from PCR-amplified fragments.
This device acts as a detector for glucose (and certain derivatives of it). The double transcription terminator ensures, that only the gene downstream of the promotor zwf is influenced by it. The zwf promotor (BBa_K1840002) is contained in the genome of Pseudomonas putida and is situated in front of the enzyme zwf (glucose 6-phosphate dehydrogenase). The repressor HexR can bind the zwf promotor sequence and thus prevent the transcription of the zwf gene. In case the glucose derivative 2-Keto-3-deoxy-6-phosphogluconate is present, it binds to the promotor and leads to a subsequent conformational change. Hence, the repressor can no longer bind and the gene(s) downstream can be transcribed. In our device, the downstream gene is mCherry, codon optimized for Pseudomonas (BBa_K1840004). Therefore, in the presence of glucose, mCherry is expressed. In various experiments, we could see that this device is in deed working.
As promotor sequence, we assumed all DNA in front of the zwf gene up to the next gene, coding for the repressor HexR. Since it is encoded on the opposite strand, there is the possibility, that zwf functions as a promotor for this gene as well. Therefore the double terminator was needed.
The double terminator was already registered in the iGEM catalogue. The zwf promotor comes from the genome of Pseudomonas putida. The mCherry is codon optimized for expression in Pseudomonas.
The part contains CpxR with an upstream arabinose-inducible promoter.
The arabinose had to be upstream of the CpxR. Also, the sequence had to be tested because of a mutation, and it works perfectly.
The CpxR comes from the genome of E coli, and the arabinose promoter comes from a biobrick.
==Part Description== This is a composite part made from Green Fluorescent Protein (GFP) and a double terminator. Green Fluorescent protein (originally from part [http://partsregistry.org/Part:BBa_E0040 BBa_E0040]) that will glow green when excited by the correct wavelengths and thus can be used as a reporter gene. This part is directly fused to a terminator (originally from part [http://partsregistry.org/Part:BBa_B0014 BBa_B0014]) so it is ideal to be fused at the end of your construct. ==Table of Statistics== {| style="color:white; background-color:#0066CC;" cellpadding="20" cellspacing="0" border="2" !Chassis !Monomer/Multimer !Aminoacid Length/Weight !Localisation !Structure !Excitation wavelengths !Emmision wavelengths |- |Prokaryotic and Eukaryotic |Bipartite |274 aminoacids | |beta-barrel(for GFP) hairpins for Terminator |501nm |511nm |} ==References== <biblio> #Valdivia pmid=8707053 </biblio>
No additional design considerations. The parts were fused together following RFC10 assembly.
GFP originates from jellyfish Aequeora victoria. For more details visit the [http://www.expasy.ch/uniprot/P42212 SwissProt entry]. The double terminator was designed by Reshma Shetty. For more details visis the [http://partsregistry.org/Part:BBa_B0014 original part page].
Double terminator consisting of BBa_B0012 and BBa_B0011
Device expressing the Gb3 Synthase enzyme under control of a constitutive promoter
A constitutive promoter was added to the sequence
The iGEM Team Panama 2011 have to design and assemble the ends of an expression platform in order to bring up by rational designing a fuctional test platform for the expression of our Team favorite BioBrick part BBa_K653000 A.K. The Biosurfactator. We choose to assembly two existing BioBrick parts from the shop, we mean The Registry. Those parts are: a GFP reporter BBa_E0040 and a double terminator BBa_B0015. We assemble them to bring up a new composite part that will serve as a well characterized ends for a expression platform for several coding sequences like our RhlAB_BBa_K653000
This GFP part was derived from the reporter device used by Elowitz's repressilator : GFPmut3* has a couple of mutations from wtGFP - S2R and S65G and S72A. Emerald and EGFP have S65T and multiple other mutations. The S2R mutation should have no effect. The Double terminator consisting of BBa_B0010 and BBa_B0012. This is the most commonly used terminator. It seems to be reliable. The assembling of this two parts was performed using the 3A Assembly Method.
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Double terminator with two copies of <bb_part>BBa_B0010</bb_part>
Modification of biobricks part BBa_B0015