- #Serial cloner how to
- #Serial cloner serial
- #Serial cloner software
ideal to study gene function under endogenous promoter, normal stoichiometry. Shows relative position (arrangement and direction). Always check the sequence! Make Research Easy. #Serial cloner software
Software to read/construct vector maps and edit plasmid sequences
#Serial cloner serial
Serial Cloner VectorFriends pDRAW PlasMapper SnapGene APE (a plasmid editor) Vector NTI DNAstrider Geneious #Build a constrcuct serial cloner software# Insert cargo into the plasmid and verify the insert sequence accuracy Other tools: NEB cutter Addgene Analyze Sequenceġ.
MCS – restriction sites OR recombination regions 5’ and 3’ Primer sites for sequence verificationĢ. Insert plasmid into cells, enable the plasmid to replicate inside the host, & select for cells carrying the plasmid Backbone compatible with cloning method. Selection marker and/or screening marker. Promoter (constitutive or inducible) operator, terminator. Ribosome Binding Site, start codon, stop codonĥ. Promote proper folding of nascent protein Uses Taq and/or topoisomerase to ligate PCR fragments into vectors Seamless requires PCR extension to add Type IIs sites to insert ends. Translation Initiation: Ribosome Binding to mRNAĭetect target protein (common Ab epitope tags) HIS4, Zeocin Auxotrophy: URA3, TRP1, LEU2, HIS3įluorescent Proteins (GFP, mCherry) LuciferaseĬonstitutive: CMV, SV40, EF1a, CAG Inducible: Tet Tissue-specific for in vivo work: varied Origin of replication determines copy number in bacteria more is not always better!į1 origin of replication for single-stranded plasmid DNA (optional)Įukaryotic expression vectors use viral ORIĪmpicillin, Kanamycin, tetracycline, chloramphenicol Easy to shuttle inserts, but more expensive & longer set-up time.Ĭommonly used methods to introduce expression construct (plasmid) Requires flanking regions recognized by site-specific recombinase. Popular Expression Vectors for mammalian cellsĬase Study 1: Optimized Vectors for CRISPR/Cas9 genome editing Goal: optimize a lentiviral backbone (lentiCRISPRv1) to produce high-titer virus for human & mouse cells Step 1 Modifications: coli: pET system HIGH expression of target protein Tags can be removed at cleavage sites place between target ORF and tag: Enzyme Maltose-binding protein (MBP) glutathione-S-transferase (GST) Human- codon optimize NLS and P2A bicistronic linker sequences. Result: 10-fold increase in functional viral titer 
Rearrange components: reposition the U6driven sgRNA cassette Step 2 Modifications.Split components into two vectors delivered with different antibiotic selection markers: lentiCas9-Blast and lentiGuide-Puro.Result:100-fold increase in functional viral titer Reference: Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Result: significant increase in crt protein and butyrate production Result: faster cell growth, similar or slightly lower butyrate production Result: only slightly less acetate & more butyrate Replace Pta with butyrate pathway genes to divert flux from acetylCoA-acetate conversion.Integrate 8 genes via DC/SC-HR Strain B4 Modifications.Adjust distance between RBS and start codon of crt gene Strain B3 Modifications.Ģ014 Aug 11(8):783-4.Ĭase Study 2: Optimizing Biosynthetic Pathways in Bacterial Cell Factories Goal: optimize conversion of CO2 to butyrate in C. Reference: Ueki T, Nevin KP, Woodard TL, Lovley DR.
#Serial cloner how to
How to optimize protein expression Remember: optimize ≠ maximize more is not always better! Converting Carbon Dioxide to Butyrate with an Engineered Strain of Clostridium ljungdahlii MBio. #Build a constrcuct serial cloner how to# Choose/Reorder ORI, promoter, solubilization tags.codon optimization and/or protein design Optimize the Host.Platform (species) and strain Optimize your Methods.transformation, selection, growth, induction, purification.#Build a constrcuct serial cloner software#.#Build a constrcuct serial cloner how to#.
0 kommentar(er)
