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Team:WITS-CSIR SA/Project/Parts
From 2011.igem.org
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Parts Submitted
Click on the part number to go to the associated registry page. Click on the part name for a brief description of the part.
This DNA part will encode for an RNA riboswitch senstive to theophylline. The RBS is not exposed in the absence of theophylline. When theophylline is present, it binds to the riboswitch and causes a conformational change which leads to the exposure of the RBS and consequently translation of the adjoining gene. In this case, the gene which will be expressed is CheZ, which is a protein fundamental to bacterial movement. This theophylline riboswitch (type1) CheZ fusion BioBrick activates the expression of the CheZ gene in a theophylline-dependent fashion. It is composed of a theophylline-sensitive riboswitch clone 8.1 (Topp and Gallivan JACS 2007; BBa_K249026 and BBa_K411001) that is detached from its associated translation unit (coding region) and fused to a CheZ gene which has a N-fusion prefix and lacks a stop codon (although a TAG stop codon is provided by the RFC 25 suffix). The riboswitch regulated N-terminal part can be fused to a reporter or other CDS.
CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case, theophylline).
This DNA part will encode for an RNA riboswitch senstive to theophylline. When no theophylline is present, the RBS within the riboswitch sequence is not exposed to translation machinery. When theophylline is present, it binds to the riboswitch and causes a conformational change which results in the RBS being exposed. This allows for the translation of the adjoining gene. In this case, the gene which will be expressed is CheZ - a fundamental protein in the signalling cascade of bacterial chemotaxis.
This theophylline riboswitch 2- CheZ fusion BioBrick activates the expression of the CheZ gene in a theophylline-dependent fashion. It consists of a newly improved theophylline riboswitch clone 12.1 (Lynch and Gallivan NAR 2009) that is detached from its associated translation unit (coding region) and fused to a CheZ gene which has a N-fusion prefix and lacks a stop codon (although a TAG stop codon is provided by the RFC 25 suffix). The riboswitch regulated N-terminal part can be fused to a reporter or other CDS.
CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case, theophylline).
When no atrazine is present, the RBS within the riboswitch sequence is not exposed to translation machinery. When atrazine is present, it binds to the riboswitch and causes a conformational change which results in the RBS being exposed. This allows for the translation of the adjoining gene. In this case, the gene which will be expressed is CheZ - a fundamental protein in the signalling cascade of bacterial chemotaxis. This riboswitch-CheZ fusion BioBrick regulates the expression of the CheZ gene in an atrazine-dependent fashion. It is composed of an atrazine-sensitive riboswitch, developed by Sinha et al (2010), which is detached from its associated translation unit (coding region) and fused to a CheZ gene which has the Freiburg N-fusion prefix and lacks a stop codon. While it is possible to fuse the riboswitch to the CheZ coding region by standard BioBrick assembly techniques, this approach was not used as it would increase the distance between the RBS and the ATG start codon and potentially decrease the efficiency of the riboswitch. This was shown in the work of the Taipei 2010 team who used this approach for a theophylline riboswitch. The riboswitch and the adjacent CheZ coding region are considered together and should be cloned together. CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the Chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case atrazine).
This part regulates the expression of venus in a theophylline-dependent fashion through an RNA aptamer specific to theophylline. This theophylline riboswitch 1 biobrick activates the expression of the Venus fluorescent protein in a theophylline-dependent fashion. It is composed of a theophylline sensitive riboswitch clone 8.1 (Topp and Gallivan JACS 2007; BBa_K249026 and BBa_K411001) fused upstream to an adjacent Venus coding region.
This is a theophylline riboswitch clone 12.1 (Lynch and Gallivan NAR 2009) fusion with the Venus reporter BBa_K354002. This part regulates the expression of venus in a substrate-dependent fashion through an RNA aptamer specific to the antibiotic theophylline.
This BioBrick represents a C-terminal fusion part. The part contains a standard suffix and a “assembly standard 25” prefix (Freiburg Fusions/ RFC 25). This version of the mRFP reporter (BBa_E1010) has the standard BioBrick suffix and prefix. The standard Biobrick prefix, however, is followed by another restriction site – specific for NgoMIV (which forms the Freiburg fusion prefix). The part lacks an ATG start codon (provided by N-terminal part). It can therefore be fused to any gene in a modular fashion via the modular construction strategy of protein fusion developed by the Freiburg 2007 team.
This BioBrick represents a C-terminal fusion part. The part contains a standard suffix and a “assembly standard 25” prefix (Freiburg Fusions/ RFC 25). This version of the Venus Fluorescent reporter (BBa_K354002) has the standard BioBrick suffix and prefix. The standard Biobrick prefix, however, is followed by another restriction site – specific for NgoMIV (which forms the Freiburg fusion prefix). The part lacks an ATG start codon (provided by N-terminal part). It can therefore be fused to any gene in a modular fashion via the modular construction strategy of protein fusion developed by the Freiburg 2007 team.
The RBS CheZ- Fusion BioBrick consists of a RBS fused to the E. coli CheZ motility factor that lacks a stop codon at the N-terminus (although a TAG stop codon is provided by the RFC 25 suffix). CheZ in this part contains the standard BioBrick prefix and the Freiburg (2007) fusion suffix. The N-terminal part can be fused to a reporter or other CDS. Using this BioBrick, CheZ is constitutively expressed.
CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling).
This part consists of an atrazine-sensitive riboswitch, developed by Sinha et al (2010), detached from its associated translation unit (coding region) and fused to mRFP1 fluorescent reporter (BBa_J61100). This part regulates the expression of mRFP in an atrazine-dependent fashion through an atrazine-specific RNA aptamer.
This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 1 (Topp and Gallivan JACS, 2007) which is fused to the Venus fluorescent reporter protein without a stop codon in between. The theophylline riboswitch1-venus fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).
This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 2 (Lynch and Gallivan NAR 2009) which is fused to the Venus fluorescent reporter protein without a stop codon in between. The theophylline riboswitch2-venus fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).
This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 1 (Topp and Gallivan JACS 2007) which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The theophylline riboswitch1-CheZ fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).
This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 2 (Lynch and Gallivan NAR 2009) which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The theophylline riboswitch2-CheZ fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).
This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an RBS which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The RBS-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015)
This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an atrazine riboswitch riboswitch (Sinha et al, 2010) which is fused to the monomeric Red fluorescent reporter protein without a stop codon in between. The atrazine riboswitch-mRFP fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).
This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an atrazine riboswitch riboswitch (Sinha et al, 2010) which is fused to CheZ and monomeric Red fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The atrazine riboswitch-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).
This composite BioBrick begins with a strong, constitutively active promoter of This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an RBS which is fused to CheZ and monomeric Red fluorescent reporter protein (mRFP) without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The RBS-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).
lox66 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularily in mouse genetics. lox site recombination is catalysed by a Site specific recombinase, Cre. lox sequences are composed of an 8 bp Core sequence surrounded by two Arms. The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox66 and a lox71 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox66 & lox71 sites is potentially interresting when the recombination reaction must be "irreversible".
Lox71 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularly in mouse genetics. lox site recombination is catalysed by a Site specific recombinase, Cre. lox sequences are composed of an 8 bp Core sequence surrounded by two Arms. The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox71 and a lox66 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox71 & lox66 sites is potentially interesting when the recombination reaction must be "irreversible".
