User:SKaret/Notebook/GroupProj/PromRes

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Factors affecting output from genetic gene circuits:

• Choice of transcription factor
• Choice of core promoter
• Operator sites
  • Number of sites
  • Spacing between
  • Binding affinity
• Transgene copy number
• Integration into the genome or expression from plasmids
• Promoter activity
• Ribosome-binding sites
• Codon bias of the host
• Transcription rate and tRNA abundance
• Half-life of mRNA
• Substrate and cofactor availability
• Adjustment of enzyme kinetics
• Protein scaffolding
• Sub-cellular localization through the use of microcompartments

These affect expression dynamics, namely strength, leakiness and sensitivity of a promoter

Constitutive promoters:

• Desirable features: different and predictable activation characteristics.
• Problems:
  • Few sequences available
  • Do not perform as required
  • Not transferable to new chassis

Inducible promoters:

• Concentration of inducer can be changed to achieve desired protein production levels
• Problems:
  • Promoter hypersensitivity
  • Cost (industrial)
  • Heterogenous expression levels
    • Variations in transgene expression levels, even in clonal populations

Structure of the prokaryotic promoter (E. Coli)

• Red: Transcription start site (TSS)
• Blue: Conserved hexamers
  • 10 and 35bp upstream of TSS
  • Key binding sites for RNA Polymerase
  • In some species a consensus length of 17bp between the conserved hexamer sequences is found
• Green: UP elements
  • Adenine/thymine rich
  • Boosts transcription rate by interacting with the C-terminal domain on the a subunit of the RNA polymerase
• KEY:
  • N = any deoxynucleotide
  • W = Adenine or Thymine
  • A = Adenine
  • T = Thymine
  • C = Cytosine
  • G = Guanine

Design principles for a synthetic promoter

1. Number of spacing between response elements:

• Transcription increases with number of operator sites 5’ of the minimal promoter.
• Spacing between response elements, and between response elements + minimal promoter increases steric hindrance
  • Changes orientation of bound TF to the minimal promoter
  • Steric hindrance = slowing of chemical reactions due to bulk

2. Variants of response elements

• Modified to increase or decrease binding affinity of TF to their cognate sequence
  • Enhanced binding --> increase transcription initiation
  • Reduced binding --> decrease transcription initiation

3. Choice of minimal promoter

• Modifies efficiency of RNA polymerase recruitment (to TSS)
• Influences basal and maximal transgene expression

4. Choice of reporter gene

• Allows quantification of the upstream promoter
  • Intracellular proteins: Faster turnover rates  Lower basal levels
  • Secreted proteins: monitor gene expression over time

Previous years project: future directions:

• Parts containing B0032/34 RBS and corresponding plasmids had mutations or missing DNA sequences
• Implement the toggle switch in the lab and carry out stability and robustness testing
• Application; Production of essential oils for use in fragrances via metabolic engineering