Our plasmid starts with a constitutive promoter upstream of a MerR protein, which is then followed by a PcadA promoter (BBa_K1724000) and Red Fluorescence Protein (BBa_I13521). The MerR protein represses the PcadA promoter in normal conditions. However, when cadmium is present, the MerR protein is released from the PcadA promoter site, allowing RFP to be expressed, signifying the presence of Cadmium.
Enzymes Start: EcoR1 and Dpn1 (Not that effective) Enzymes End: Pst1 and Spe1
(BBa_K3466001) Modified Construct 1 with the addition of one piece of junk DNA as a spacer before the cadmium sensitive promoter, PcadA.
Enzymes Start: EcoR1 and Dpn1 (Not that effective) Enzymes End: Pst1 and Spe1
Modified Construct 1 with one piece of junk DNA as a spacer after the cadmium sensitive promoter, PcadA.
Enzymes Start: EcoR1 and Dpn1 (Not that effective) Enzymes End: Pst1 and Spe1
Modified Construct 1 with the addition of BamHI and BgIII restriction sites surrounding the RBS. BamHI “BamHI (pronounced "Bam H one") (from Bacillus amyloliquefaciens) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site.” BgIII “Thermo Scientific BglII restriction enzyme recognizes A^GATCT sites and cuts best at 37°C in buffer O. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.”
Enzymes Start: EcoR1 and Dpn1 (Not that effective)
Enzymes End: EcoRI 1 and Spe1
Modified Construct 2 with the addition of BamHI and BgIII restriction sites surrounding the RBS.
BamHI
“BamHI (pronounced "Bam H one") (from Bacillus amyloliquefaciens) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site.”
BgIII
“Thermo Scientific BglII restriction enzyme recognizes A^GATCT sites and cuts best at 37°C in buffer O. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.”
Modified Construct 3 with the addition of BamHI and BgIII restriction sites surrounding the RBS.
BamHI
“BamHI (pronounced "Bam H one") (from Bacillus amyloliquefaciens) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site.”
BgIII
“Thermo Scientific BglII restriction enzyme recognizes AGATCT sites and cuts best at 37°C in buffer O. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.”
Construct 7 is an alternate version of Construct 1 with the substitution of a T7 promoter. This should increase efficiency in our cell-free system.
This construct will be useful to us as a positive control when testing the RFP expression of the other constructs once a transformation is carried out. Therefore, it was included in our order of plasmids.
Plasmid Backbone pSB1C3: Chloramphenicol (CAM) resistance.
Enzymes EcoR1 and Spe1
J6011: RBS binding site. Comes from a similar family of RBS binding sites found from a saturation mutagenic library.
K172400: Cadmium sensitive promoter. CadA promoter is a cadmium-sensitive promoter. Its repressor is MerR. In high concentration of cadmium, the Cd2+ can rapidly bind with MerR and remove its inhibition of CadA promoter. The reverse is the opposition.
K608010: Medium constitutive promoter with strong RBS and GFP.
K142000 lacI IS mutant E.Coli. The lacI IS mutant is almost identical to the lacI transcriptional regulator except for the difference that it is not able to bind IPTG or allolactose due to a mutation. It can not be activated with these substances, but since it recognizes the same motif in the lac promoter region, it strongly represses transcription of all genes regulated by promoters with lacI binding site even if IPTG or allolactose are present.
B0030: Strong RBS for E.Coli. It has a global non-modularity towards promoters & protein coding parts and relative strength was estimated for RBSs B0030, B0032, B0034. It is not compatible with R0053 promoter due to likely secondary structure transcription.
E0040: GFP. GFP is a green fluorescent protein derived from the jellyfish Aequeora victoria. It can be used to test the strength of promoters or as a reporter gene depending on the fluorescence intensity.
J61100: RBS binding site. Belongs to a family of similar ribosome binding site identified from a saturation mutagenic library.
K346002: PmerT promoter comes from Tn21 mercury resistance (mer) operon. It responds to mercury concentrations with a dose-response.
J33203: E. coli autonomous replication system promoter, arsR gene and lacZ
B0030: “Strong RBS based on Ron Weiss thesis. Strength is considered relative to BBa_B0031, BBa_B0032, BBa_B0033 and BBa_B0034”
K142000: Mutation of a transcription regulator that doesn’t diffuse in contact with allolactose or IPTG.
Construct 5
Construct 6
Construct 7
RBS+mRFP
Parts: