37℃ induced RBS + tetR + double terminator
It is a transcriptional unit including 37℃ induced RBS + tetR + double terminator. It can be added various promoter to adapt your conditions.
To regulate following genes by temperature. To maintain cells at 37?? C ↑ : the tetR protein be generated and repress Ptet's circuit. To release cells at 37?? C ↓ : the tetR protein not be generated and do not repress Ptet's circuit.
the 37℃ induced RBS cames from iGEM08_TUDelft
Quorum sensing is the mechanism of communication between bacteria via synthesizing and detecting chemical compounds in the media. With these mediators, the promoters and their yield can be tuned or regulated. This part represents an alternative quorum sensing sender device which is designed to induce PoPS production in the parts including pLas such as BBa_K772004. LasI is an enzyme generator gene sequences which synthesizes 6HSLO3 (AHL) in order to induce a PoPS response in the pLas promoter. pLas promoter works in the presence of both 6HSLO3 and LasR protein.
Note that there is an RBS sequence just before LasI; in order to be easily used with quorum sensing receiver devices such as BBa_F2620 or BBa_K772004.
LasI sequence is gathered from Registry.
To edit. Pense ?? rajouter les linkers, je ne sais pas s'ils sont de igem ou s'il faut ajouter la s??quence ?? la registry. Check pour tous tes biobricks et dis-moi, please.
AHL regulated PoPS generator: Las receiver device
Quorum sensing is the mechanism of communication between bacteria via synthesizing and detecting chemical compounds in the media. With these mediators, the promoters and their yield can be tuned or regulated. This part represents an alternative quorum sensing receiver device, which is able to generate a PoPS response in the presence of 6HSLO3 produced by LasI, instead of BBa_F2620. This will allow iGEMers to establish communications between different bacteria colonies and it makes simple to generate easy regulated complex systems. This part is assembled with Las promoter system to improve the current quorum sensing devices and parts which commonly base on Lux promoter system.
Note that pLas has no RBS afterwards, the assembling methods must be selected after this consideration.
pLas and LasR sequences are gathered from Registry.
The IbpB thermometer is an RNA thermometer element found in the ibpAB operon. The operon contains two heat-shock genes, encoding inclusion body binding proteins A and B (IbpA/B), and is the most drastically upregulated operon under heat-shock in Escherichia coli. IbpA is regulated by a ROSE element found in its 5' UTR, while IbpB has its own heat-sensitive cis-regulatory element. The activity of this thermoregulator was confirmed in vitro but was not found in vivo, suggesting more complicated operon regulation exists in bacterial cells. In this part, red fluorescent protein (RFP) is added in the downstream region to measure the activity of RNA thermometer. You check our Experience page for further information about our characterization and experiments. For the inner elements in the part design; check out the Part Design page. RNA Thermometers are the only known single-component regulators of gene expression.(1) RNA Thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Naturally occurring RNA Thermometers exhibit complex secondary structures which are believed to undergo a series of gradual structural changes in response to temperature shifts.(2) RNA Thermometers often regulate genes required during either a heat shock or cold shock response, but have been implicated in other regulatory roles such as in pathogenecity and starvation.(3)
Note that RNA thermometer actually works as an RBS sequence. Therefore, this part does not include an additional ribosome binding site. Also, this part does not contain an terminator sequence, but a simple stop codon. The RFP should be translated continuously.
Escherichia coli, NCBI GeneBank Other elements are obtained from Registry.
RecA is a UV-light-inducible promoter. Trans is a "key" of sorts, acting on a complementary cis "lock" to allow the translation of ccdB, a kill gene. Together, the RecA+Trans system allows for the kill gene to be translated in the presence of UV light, through the indirect induction by way of the cis-trans system.
We had to consider whether the design was too complicated, whether it wouldn't be better to just have a more simple, direct system whereby a kill gene was translated because of the action of a UV-light-inducible promoter. In the end, we decided to make both systems, as the latter has been done before and submitted to the iGEM Registry of Standard Biological Parts.
Source parts were obtained from the iGEM Registry of Standard Biological Parts.
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.
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[1]. Biobrick BBa_K1486013 has CpxR-IFP[2] with an arabinose promoter. This construct was made in both orientations: BBa_K1486014 is IFP[1]-CpxR (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[2]. Biobrick BBa_K1486012 has CpxR-IFP[1] with an arabinose promoter. This construct was made in both orientations: BBa_K1486015 is IFP[2]-CpxR (with an arabinose promoter).
Both orientations were made, as well as for IFP[1] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
recNp, could be started with exposure to irradiation, UV, nalidixic acid
Name: recNp +1: 2749781 Sigma Factor: Sigma70 Sigmulon Distance from start of the gene: 36 Sequence: aattattctaattttacgccagcctctttactgtatataaaaccagtttatactgtacacAataacagtaatggtttttca -35 -10 +1 Note(s): We assigned a putative transcription start site to this promoter based on the observation that the majority of the promoters, whose transcription start sites were determined experimentally, present a distance of 6 nucleotides between the transcription start site and the -10 box. Evidence(s): [HIPP] Human inference of promoter position Reference(s): [1] Rostas K., et al., 1987
recAp, could be started with exposure to irradiation, UV, nalidixic acid
Name: recAp +1: 2821841 Sigma Factor: Sigma70 Sigmulon Distance from start of the gene: 50 Sequence: cttgtggcaacaatttctacaaaacacttgatactgtatgagcatacagtataattgcttCaacagaacatattgactatc Evidence(s): [HIPP] Human inference of promoter position [TIM] Transcription initiation mapping Reference(s): [1] Pages V., et al., 2003
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[1]-CpxR. Biobrick BBa_K1486015 has IFP[2]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486012 is CpxR-IFP[1] (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put between the promoter and the cds.
This part is the twin of the non-compatible biobrick BBa_K1486008 rendered compatible by mutating the two illegal restriction sites. However, all experiments were run on the aforementioned biobrick.
Site-directed mutagenesis was used to remove the restriction sites in the sequence.
http://www.addgene.org/browse/article/8177/ which was then mutated by us.
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.
araC-Pbad - Arabinose inducible regulatory promoter/repressor unit
Valentina Herbring, Sebastian Palluk, Andreas Schmidt
This promoter can be induced by arabinose. In presens of arabinose the promotor is active.
Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121-30 Khlebnikov A, Skaug T, Keasling JD, modulation of gene expression from the arabinose-inducible araBAD promoter,J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7.
Constitutive promoter+37℃ induced RBS+tetR+double terminator+Ptet+RBS+GFP+double terminator
We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???.
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.
GFP - LasR protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, an inducer regulatory protein (LasR is responsible to induce pLas promoter in the presence of 6HSLO3 which is a chemical compound sythesized by an enzyme that LasI gene codes.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.
Note that there is an RBS sequence just before the protein sequence in order to eliminate the need of addition of it.
Cleavage site, LasR and GFP sequences are gathered from Registry.
This E. coli promoter is positively regulated by arabinose. araC is the regulator protein, binding to arabinose prevents the repression of pBAD.
This part is similar to BBa_I0500, but it differs from that one in several point mutations in the sequence.
The repression of heat shock gene expression (ROSE) element is an RNA element found in the 5' UTR of some heat shock protein's mRNAs. The ROSE element is an RNA thermometer that negatively regulates heat shock gene expression. The secondary structure is thought to be altered by temperature, thus it is an RNA thermometer. This structure blocks access to the ribosome binding site at normal temperatures. During heat shock however, the structure changes freeing the ribosome binding site and allowing expression to occur. RNA Thermometers are the only known single-component regulators of gene expression.(1) RNA Thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Naturally occurring RNA Thermometers exhibit complex secondary structures which are believed to undergo a series of gradual structural changes in response to temperature shifts.(2) RNA Thermometers often regulate genes required during either a heat shock or cold shock response, but have been implicated in other regulatory roles such as in pathogenecity and starvation.(3)
Bradyrhizobium japonicum USDA 110 and Registry
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the N terminus of CpxR).
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[2]-CpxR. Biobrick BBa_K1486014 has IFP[1]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486013 is CpxR-IFP[2] (with an arabinose promoter).
Constitutive promoter + 37℃ induced RBS + tetR + double terminator
we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???. We speculate that when the ???Regulator??? and ???Production??? sections are combined, we can achieve a high bacteria growth rate and close to no target protein producing rate at T>37??C, and low bacteria growth rate and high target protein producing rate at T<37??C.
To regulate following gene by temperature
GFP - LasR anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, an inducer regulatory protein (LasR is responsible to induce pLas promoter in the presence of 6HSLO3 which is a chemical compound sythesized by an enzyme that LasI gene codes.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
Pgad - Intracellular Chloride-Sensing Cassette
Pgad is chloride-sensitive promoter which was first discovered in Lactococcus lactis1, which is a gram-positive bacterium which can live in acidic environment. In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of Pgad2. The gene before Pgad, named gadR, which is constitutively expressed under the control of PgadR, is a positive regulator of Pgad coupled genes while intracellular chloride is level elevated.
Synthesized part by GenScript,all the illegal sites for RFC 10 (EcoRI, XbaI, SpeI, PstI) have been substituted by other codon producing the same amino acid in E. coli.
The gene sequence was obtained from the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes PgadR, gadR, Pgad and the starting codon ATG.
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: - IFP[1]-CpxR + IFP[2]-CpxR (BBa_K1286009) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
Repression of heat shock gene expression (ROSE) element The repression of heat shock gene expression (ROSE) element is an RNA element found in the 5' UTR of some heat shock protein's mRNAs. The ROSE element is an RNA thermometer that negatively regulates heat shock gene expression. The secondary structure is thought to be altered by temperature, thus it is an RNA thermometer. This structure blocks access to the ribosome binding site at normal temperatures. During heat shock however, the structure changes freeing the ribosome binding site and allowing expression to occur.[2][3] A partial structure of this RNA element has been determined using NMR.[1] ROSE1 and ROSEAT2 are specific examples of ROSE elements.[4] ROSE1 is found in Bradyrhizobium japonicum whereas ROSEAT2 is a closely related element from Agrobacterium tumefaciens. The two RNA elements have similar secondary structures with ROSE1 having an extra hairpin.[4] All ROSE elements contain a characteristic 'bulged G' opposite the Shine-Dalgarno sequence binding site, without this nucleotide the RNA thermometer loses its temperature-sensitivity.[5]