IP-Free E. coli Inducible Expression Vectors E. coli expression vectors are available with the following promoters: T5 or T7 (IPTG-inducible), rhabad (rhamnose-inducible), ara (arabinose and IPTG-inducible) and phoa (induced by phosphate starvation). These vectors express in any strain of E. coli, except T7 promoter vectors which require a strain that expresses the T7 RNA polymerase and ara promoter vectors which require a strain that expresses the repressor AraC. IP-Free E. coli Expression Vectors with the IPTG-inducible T7 Promoter To vary expression levels, vectors with the T7 promoter are available with different strength ribosome binding sites and a choice of high and low copy origins of replication. These vectors are also available with a choice of resistance markers. Plasmid Map ATG Protease Cleavage Site Your Gene GGT Term_T7 P_Amp M_Affinity Tag 4 5 0 0 1 5 0 0 Term_bla 4 0 0 0 3 5 0 0 1 0 0 0 M_Ampicillin- r_* Term_rpoC 1 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 M_lacI_* P_lacI Name Affinity Tag Protease Cleavage Site Qty Storage pd454-fh8 FH8 PCS_TEV 10Rx -20 pd454-gst GST PCS_HRV3C 10Rx -20
Plasmid Map PCS_TEV ATG Your Gene GGT Term_T7 P_Amp M_PpiB 4 5 0 0 1 Term_bla Term_rpoC 3 5 0 0 4 0 0 0 pd454- PpiBT T7- PpiBT- ORF, Ecoli- ElecD 4543 bp 5 0 0 1 0 0 0 M_Ampicillin- r_* 3 0 0 0 1 5 0 0 2 5 0 0 2 0 0 0 M_lacI_* P_lacI Name Qty Storage pd454-ppibt 10Rx -20 Plasmid Map Your Gene ATG GGT Term_T7 P_Amp 5 0 0 0 1 M_MBP 4 5 0 0 5 0 0 4 0 0 0 pd454- MBP T7- MBP- ORF, Ecoli- ElecD 5122 bp M_Ampicillin- r_* 1 0 0 0 Term_bla 3 5 0 0 1 5 0 0 P_lacI Term_rpoC 3 0 0 0 2 5 0 0 2 0 0 0 M_lacI_* Name Qty Storage pd454-mbp 10Rx -20
T7 Induction Protocol The IPTG-inducible T7 promoter works in E. coli strains BL21 (DE3) or T7 Express. The promoter is followed by lac operators that are recognized by the lac repressor, which is also carried on the plasmid. IPTG induces expression by binding to the repressor. Grow cells overnight in LB plus antibiotic. Dilute into fresh LB with antibiotic, grow to mid-log (A600 0.6-0.8), induce by adding IPTG to 1 mm IPTG, and grow for an additional 4-8 hours. T7 Vector Controls Any E. coli-optimized Protein Paintbox gene in an Electra MOTHER vector can be cloned into any Electra T7 DAUGHTER vector.
Electra Cloning System Electra is a simple one-tube universal cloning process that can be performed in a 5 minute bench-top reaction with the fidelity of a restrictionbased cloning system. A gene from one MOTHER vector is compatible with all DAUGHTER vectors, allowing rapid testing of many different sequence contexts simultaneously. Reagents The Electra Reagents kit contains all necessary components to facilitate cloning a gene from a MOTHER into a DAUGHTER vector. The Electra reaction can also be used to clone a PCR product into either a MOTHER or a DAUGHTER vector. Electra Buffer Mix is supplied at 10X final concentration (use 2 µl in a 20 µl reaction) Electra Enzyme Mix is supplied at 20X final concentration (use 1 µl in a 20 µl reaction) Cloning Protocol Component Volume (µl) MOTHER DNA / Positive control (20 ng) 1 DAUGHTER Vector (20 ng) 1 Electra Buffer (10x) 2 Electra Enzyme (20x) 1 Water 15 Total 20 1. Combine components and incubate at 25-37 C for 5-20 minutes. 2. Transform 1-2 µl into chemically competent E. coli. (DH10B cells recommended) 3. Recover cells for 45 minutes and then plate on appropriate antibiotic for the DAUGHTER. 3a. Optionally include streptomycin at 100 µg/ml (for selection against pmother with rpsl); or plate on YEG with antibiotic plus p-chloro phenylalanine at 10mM (for selection against pmother with phes). Positive Control A positive control MOTHER vector carries a gene in which Ptet drives expression of green fluorescent protein (DasherGFP). A successful Electra reaction will produce green fluorescent colonies from the DAUGHTER vector. Electra DAUGHTER Vectors Electra DAUGHTER vectors are supplied as linearized DNA, with overhangs compatible with an ATG (encoding methionine) at the 5' end and GGT (encoding glycine) at the 3' end.
Electra MOTHER Vectors Genes in MOTHER vectors have adjacent restriction sites that produce overhangs compatible with an ATG at the 5' end and GGT at the 3' end upon digestion with SapI. Alternatively Electra ends can be added to any gene* by PCR. We recommend you add the following ends to your PCR primers: 5'-TACACGTACTTAGTCGCTGAAGCTCTTCTATG...(ORF)...-3' 5'-TAGGTACGAACTCGATTGACGGCTCTTCTACC...(ORF Reverse Complement)...-3' *Your gene must not contain any internal SapI recognition sites, since the Electra cloning process utilizes the typeiis enzyme SapI. MOTHER vectors also contain a counter-selection gene. This can be used to eliminate any residual gene propagating in the MOTHER. Feature list descriptions Ampicillin-r FH8 GST laci,p_laci MBP PCS_HRV3C PCS_TEV PpiB A semi-synthetic penicillin derived from 6-amino-penicillanic acid causes cell death by inhibiting cell wall biosynthesis. The gene coding for ampicillin resistance (bla) is a beta lactamase which is secreted into the periplasmic space where it catalyzes hydrolysis of the beta-lactam ring of ampicillin. E.coli transformed with plasmid containing the ampicillin resistance gene can grow on media containing 50-100 µg/ml ampicillin. (www.jac.oxfordjournals.org/content/43/5/699.full) A 8 kda solubility tag that enhances solubility. It can serve as an affinity tag and binds to hydrophobic columns. (http://www.ncbi.nlm.nih.gov/ pubmed/23160981) Glutathione S-transferase (GST) is a naturally occurring 26 KDa protein found in eukaryotic cells. It is used as an affinity tag to aid in the purification of recombinant proteins. GST as a fusion tag can act as a chaperone to facilitate protein folding. The GST moiety binds with high affinity to glutathione coupled to a sepharose matrix and the fusion protein can be eluted under mild, non-denaturing conditions using reduced glutathione. The GST affinity tag can be removed by using a site-specific protease recognition sequence located between the GST moiety and the target sequence. (www.ncbi.nlm.nih.gov/pmc/articles/pmc3584333/) laci is a regulatory gene of the lac operon that codes for the repressor that binds very tightly to a short DNA sequence just downstream of the promoter near the beginning of lacz called the lac operator. The repressor binding to the operator interferes with binding of RNAP to the promoter, and therefore transcription occurs only at very low levels. (www.en.wikipedia.org/wiki/lac_repressor) LacO is a regulatory gene of the lac operon. If lactose is missing from the growth medium, the repressor binds very tightly to a short DNA sequence just downstream of the promoter near the beginning of lacz called the lac operator. The repressor binding to the operator interferes with binding of RNAP to the promoter, and therefore transcription occurs only at very low levels. When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose, which is a combination of glucose and galactose, binds to the repressor, causing a change in its shape. Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe and thereby leading to higher levels of the encoded proteins. Silencing of the promoter prior to IPTG induction is achieved using symmetrical lac operators (Proc Natl Acad Sci USA 1983. 80:6785. Sadler et al) spaced around the promoter to maximize cooperativity (EMBO J 1994. 13:3348. Oehler et al). This operator pair ensures significantly tighter repression than regular lac operators. Overlapping T5 promoter/lac operator has been described (Proc Natl Acad Sci USA 1988. 85:8973. Lanzer and Bujard ). (www.ncbi.nlm.nih.gov/pubmed/6316325) Maltose binding protein (MBP) is a commonly used protein expression tag for tagging proteins expressed in microbes and is also known to significantly enhance solubility of many proteins. Fusion of a target protein to MBP permits its one-step affinity purification using amylose-agarose resin. (http://www.ncbi.nlm.nih.gov/pubmed/20978971) The origin of replication is a sequence in a genome at which replication is initiated. The puc ori is a mutated form of origin derived from E. coli plasmid pbr322 which allows production of greater than 500 copies of plasmid per cell. (www.en.wikipedia.org/wiki/origin_of_replication) A phage T7 derived strong promoter which is recognized by T7 RNA polymerase. The promoter is controlled by a lac operator sequence that allows induction by addition of IPTG. A protease cleavage site recognized by Human rhinovirus 3C protease (HRV3C Protease) which is a cysteine protease that recognizes the cleavage site of Leu-Glu-Val-Leu-Phe-Gln*Gly-Pro, and cleaves between the Gln and Gly residues. HRV3C protease is used to remove fusion tag from proteins with the HRV 3C cleavage sequence. (http://www.jbc.org/content/274/19/13211.full) TEV protease for bacterial expression from pcs plasmid. (www.addgene.org/empty_backbones/) A stress-responsive protein when used as a fusion partner acts as a solubility enhancer. (www.onlinelibrary.wiley.com/doi/10.1111/ j.1574-6968.2009.01616.x/pdf) A ribosome binding site (RBS) is a sequence on mrna that is bound by the ribosome during protein translation. It can be either the 5' cap of a mrna in eukaryotes, a region 6-7 nucleotides upstream of the start codon AUG in prokaryotes (called the Shine-Dalgarno sequence), or an internal ribosome entry site (IRES) in viruses. Prokaryotic ribosomes recognize RBSs primarily via base-pairing between the RBS and an unstructured end of the 16s rrna molecule that forms part of the ribosome. Translation initiation rate of a particular mrna can be regulated by sequence of the RBS, leading to varying strengths - strong, medium or weak. (www.msb.embopress.org/content/7/1/481.abstract)
Licenses DNA2.0 Catalog Product Purchases By purchasing DNA2.0 Catalog Products, Customer agrees that Customer will comply with the compliance terms set forth in Section 9 (see /company/terms-and-conditions). Electra Daughter Vectors The purchase of DNA2.0 Electra Vectors conveys to CUSTOMER the limited, non-transferable right to use the purchased amount of the linearized Electra Vector. (For clarity, once CUSTOMER's ORF of interest has been cloned into the Electra Vector, CUSTOMER may replicate the resulting plasmid.) CUSTOMER may incorporate DNA2.0 Electra Vectors into CUSTOMER'S product as specified in Section 9 (see /company/terms-and-conditions), provided that CUSTOMER does not resell the Electra Vectors or vectors derived from the Electra Vectors as standalone vectors or incorporated into cloning kits.