- #Build a constrcuct serial cloner how to#
- #Build a constrcuct serial cloner software#
transformation, selection, growth, induction, purification. Platform (species) and strain Optimize your Methods. codon optimization and/or protein design Optimize the Host. Choose/Reorder ORI, promoter, solubilization tags. #Build a constrcuct 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.
Reference: Ueki T, Nevin KP, Woodard TL, Lovley DR. 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:.
2014 Aug 11(8):783-4.Ĭase Study 2: Optimizing Biosynthetic Pathways in Bacterial Cell Factories Goal: optimize conversion of CO2 to butyrate in C. Improved vectors and genome-wide libraries for CRISPR screening. Reference: Sanjana NE, Shalem O, Zhang F. Result:100-fold increase in functional viral titer
Split components into two vectors delivered with different antibiotic selection markers: lentiCas9-Blast and lentiGuide-Puro. Rearrange components: reposition the U6driven sgRNA cassette Step 2 Modifications:. Result: 10-fold increase in functional viral titer Human- codon optimize NLS and P2A bicistronic linker sequences. 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) 
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. Uses Taq and/or topoisomerase to ligate PCR fragments into vectors Seamless requires PCR extension to add Type IIs sites to insert ends. Promote proper folding of nascent protein
Ribosome Binding Site, start codon, stop codonĥ. Promoter (constitutive or inducible) operator, terminator. Selection marker and/or screening marker. Backbone compatible with cloning method. Insert plasmid into cells, enable the plasmid to replicate inside the host, & select for cells carrying the plasmid MCS – restriction sites OR recombination regions 5’ and 3’ Primer sites for sequence verificationĢ. Insert cargo into the plasmid and verify the insert sequence accuracy Other tools: NEB cutter Addgene Analyze Sequenceġ. Serial Cloner VectorFriends pDRAW PlasMapper SnapGene APE (a plasmid editor) Vector NTI DNAstrider Geneious #Build a constrcuct serial cloner software#
Software to read/construct vector maps and edit plasmid sequences
Always check the sequence! Make Research Easy. Shows relative position (arrangement and direction). ideal to study gene function under endogenous promoter, normal stoichiometry. Targeted genome editing: CRISPR/Cas9 is simpler and more efficient than ZFN, TALEN, Cre-lox. Viral-mediated integration – untargeted. coli, transient expression in mammalian cells, or reporter assays endogenous expression Expression from plasmid DNA Goal: Knock-down endogenous gene expressionĭNA gRNA Goal: CRISPR/Cas9 genome editing Molecular Biology SpecialistĮxpression vectors: how to choose or customize vectors for gene & protein expressionĮxpression Vectors: What are they? Plasmids that carry cargo (insert DNA) into cells and allow the cargo DNA to be efficiently expressed.ĭNA shRNA DNA mRNA protein Goal: express heterologous protein Expression vectors: how to choose or customize vectors for gene & protein expression Rachel Speer, Ph.D.
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