Procaryotic Operon

Transcription

1 2 Basics of Cloning

2 Procaryotic Operon

3 Promotor Operator ATG..TAA CDS

4 Expression plasmid Required factors for protein expression

5 Promoters Constitutive promoters Always active, continuous culturing possible (e.g. combination with secretion). Disadvantage: toxic proteins inhibit biomass generation. Inducible promoters Expression is induced by physical or chemical stimulus. Production of biomass and protein are separated in time. Disadvantage: continuous culturing impossible, potentially costly inducer.

6 Required factors for protein expression Origin of replication (ORI) Enables independent replication of the plasmid. Ideally at high copy number. The choice of ORI may influence expression strenght. The F1 origin is used for historic reasons, when single stranded plasmids were required for DNA sequencing.

7 Required factors for protein expression? Selectionmarker Since the mainenance of a plasmid requires large amounts of energy, the bacterium will try to get rid of it. A selection marker offers a benefit to the bacterium (e.g. resistance against an antibiotic, or an alternative source of nutrients). A selection pressure towards plasmid mainenance is created. Common selection markers: ß-Lactamase (Ampicillin-resistance), N-Acetyltransferase or Phosphotransferase (Kanamycin-resistance)

8 Required factors for protein expression? Repressor (optional) Prevents uncontrolled expression in the absence of an inducer.

9 Promoter systems lac-operon Strong expression, option for autoinduction, leaky. ara-operon Strong expression at stronger control. tet-operon Lower expression level, but tightly controled. tac-promoter Hybrid of the constitutive trp-promoter and the lacoperator. Strong expression, tends to be leaky. T7-Promoter Very strong expression, requires T7 RNA polymerase, usually combined with lac-operator

10 Cloning? Cloning is usually referring to the generation of identical copies of (multicellular) organisms from a somatic (differentiated) cell. The term Molecular Cloning refers to the generation of multiple copies of mostly recombinant DNA. Cloning is thereby here defined as the re-arrangement of DNA (for the purpose of protein expression) with all steps to be taken: Isolation of genetic material Amplification and modification Assembly of vectors Amplification, use Extraction, Gene synthesis PCR Restriction, ligation, recombination Transformation, selection, extraction

11 PCR

12 Primerdesign Denaturation: C Annealing: C Extension: C T m target area To be avoided: Primer-dimers Hairpin-loops Self-complementarity 5 - Attachment spec. primer Useful attachments: Restriction sites Recombination sites Affinity-Tags Protease-recognition sites Ribosome-binding sites

13 Exkurs: Attachments 5 -Phosphate: Ligation in dephosphorylated vector Generation of single stranded DNA with -exonuclease Radioaktive labelling with 32 P 5 -Biotin: Purification of PCR-products (not suitable for ligation) 5 -Fluorescein: Marker in gels 5 -Thiol- oder Aminomodifier: Surface immobilization dutp: Reduction of cross-contamination (analytical PCR)

14 Access to genetic material Eukaryots cdna synthesis (complementary DNA) mrna AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA cdna Hybridization with oligo-dt Elongation with reverse transcriptase TTTTTTTTT TTTTTTTTT AAAAAAAA ds-cdna optional: second strand synthesis

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16 Gene synthesis Gene synthesis Computation reduces faulty assembly 48 oligos 921 bp 2 oligos 80 bp

17 Design of gene synthesis

18 Codon-choice is not random aa Codon A. thali- Homo S. cere- E. coli ana sapiens visiae Why Gene Synthesis? The genetic code is universal, but... Ala GCG GCA GCT GCC Arg AGG AGA CGG CGA CGT CGC Leu TTG TTA CTG CTA CTT CTC Codons vs. 20 AS 18 AS use synonymous codons Alternative codons are being used with different frequencies Codon-Usage differs in (almost) all organisms frequent rare

19 Exkurs: Isothermal Amplification General Principle: Strand Displacement Polymerization Source:

20 Loop-mediated isothermal amplification Source: