Multi sensor - for calculation of specific concentrations of nitrates nitrites and nitric oxide
A serious problem encountered again and again by synthetic biologists is, that for a particular ligand specific promoters do not exist and that it is very difficult to construct a transcription factor that is specific to the required ligand. There are however often broad spectrum (non-specific) promoters for a ligand of interest. These promoters and their transcription factors will induce transcription initiation when exposed to (or not exposed to) the investigated ligand, but also when under influence of other similar ligands. Assuming competitive binding, this is an interesting effect which can be exploited to give specific and accurate concentrations of each of the inducing ligands. In its simplest form of the Multi-Sensor system, there are two different transcription factors, each of which will cause transcription when exposed to either one or both of two different competitive ligands. They both posses different active sites, which causes there to be a different bias in both of those for each ligand. This will lead to transcription rates of one of the promoters to indicate a ratio between the two different ligands (for example nitrates and nitrites) as seen in line 1 ("Figure 3."). Because the two different transcription factors have different binding efficiencies to the two ligands, the line of the other promoter will take a different angle (line 2). The point where these two lines cross, gives the precise concentration of both ligands even though the two different promoters are non-specific. This effect can be modelled for more than two substrates. Visually the system can be designed with each concentration being a different access on a graph (which can soon become hyper dimensional). When you add a third ligand it will result in the two lines in Figure 5. to become two planes which intersect along one line. This means that a third promoter and transcription factor is necessary (the same way that two promoters pinpoint any single point in two-dimensional space, three are needed to triangulate a point in three-dimensional space). Once the third promoter and transcription factor is added to the graph, the three planes created intersect at a single point which gives the specific concentration of each of the ligands (Figure 5:). When four ligands are used, a hyper dimensional graph of four spatial dimensions with four different plains, each pertaining to a single promoter and transcription factor, will all intersect at a single point. This point will give the specific concentration of each of the four ligands (and so on and so on). Functional construction There are a number of different ways in which this theory could be put into practice. One would involve each one of the different promoters selected to be be fused to a fluorescent protein and cloned into different cells. In addition a constitutively expressed fluorescent protein will be used to control for metabolic and cell mass differences. A homogenised sample, split between each of the culture media, containing fluorescent protein with different promoters and The expression of each fluorescent protein will be measured using fluorometer and the data is to be mathematically analysed (see below). This will give the exact concentrations of each of the ligands. Another possible way of constructing the Multi-sensor system, would be to ligate all promoter - fluorescent sequences into one plasmid and transform this into a single cell. At this point the constitutively synthesised fluorescent protein for control, is no longer necessary because the comparison is made between expressions of different proteins within the same cell. This system has advantages and disadvantages. Because each cell contains the all promoters necessary for the system, each single cell can give a reading for the chemical concentrations of the ligands investigated. This means in its direct vicinity, that each cell can become a single ??? pixel???. This would allow can for an image of chemical concentrations throughout a sample. The useful applications are vast. For example, in environments where diffusion rate is low and chemical concentrations vary e.g. soil, the multi-sensor system would be able to give accurate and locally precise data. It enables exact measurements of concentrations via laser microscopy but an overview of a great amount of data would also be available with just a simple photograph.
nir promoter from Synechocystis sp. PCC6803
The ''nirA'' promoter originates from ''Synechocystis'' sp. PCC6803.
The ''nir'' promoter is nitrate inducible.
Weak Ribosome Binding Site (BBa_B0032) plus GFP reporter with LVA degradation tag and transcription terminators.
construction intermediate in the creation of a Self Destructive Plasmid
Part was synthesized based on the promoter of the nar operon naturally present in E. coli.
The narG promoter is located at the beginning the nar operon and is naturally activated during nitrogen respiration of E. Coli by the Fnr protein to signal reduction of nitrate to nitrite. In the presence of nitrate, Fnr protein binds to a region upstream of narGp to act as an activator.
Design includes BioBrick suffixes and prefixes, and the promoter sequence itself was checked for restriction digest sites to ensure compliance with Assembly Standard 10.
RBS + highly engineered mutant of red fluorescent protein from Discosoma striata (coral) + double terminator
Mammalian-Bacterial promoter with eCFP reporter: CArG promoter sequence + BBaK216005 + BBa_E0420
E9-ns2 CArG promoter sequence + BHN + BBaK216005
Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , in the orientation mammalian to bacterial, both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably. The hybrid promoter has been attached to the reporter: enhanced Cyan Fluorescence Protein (eCFP). The hybrid promoter has been characterised by observing expression of flourescent protein, and found to have increased transcription in response to increasing concentrations of potassium nitrate.
This biobrick contains the RBS and terminators, meaning we can simply add the promoter of choice to complete the system.
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.
pJBA111, carrying GFP-LVA, was generously provided by Dr. S. Molin, The Technical University of Denmark, Lyngby Appl Environ Microbiol. 1998 Jun;64(6):2240-6. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S. PMID: 9603842
Green Fluorescent Protein with LVA tag for rapid degradation
Designed out of pJBA111 (Molin, 1998) with PCR to add appropriote prefix and suffix.
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
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.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
Double terminator consisting of BBa_B0012 and BBa_B0011
Mammalian-Bacterial Promoter: E9-ns2 CArG promoter sequence + BHN + BBaK216005
PyeaR promoter (BBaK216005) and the E9-ns2 CArG promoter (Ref1) Ref1: Scott, S.D., Joiner, M.C. & Marples, B., 2002. Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy. Gene therapy, 9(20), p.1396-402. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12365005 [Accessed June 24, 2012].
A hybrid of the PyeaR promoter (BBaK216005) and the E9-ns2 CArG promoter (Ref1). Two versions will be synthesised, with the promoters altering their position in relation to the 5???-end of the sequence.
To provide additional restriction enzyme sites that may become useful during later cloning steps, BamHI, HindIII and NdeI have been added between the 2 promoters. It is envisaged that any open reading frame (e.g. RFP or GFP) will be cloned ???downstream??? (i.e. at the 3???-end) of these promoter sequences.
Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>.
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.
engineered cyan fluorescent protein derived from A. victoria GFP
Conversion of the iGEM parts registry to SBOL2.1