User:SKaret/Notebook/GroupProj/PromoterEngineering

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Promoters

Background:

• Promoters modulate transcription and modulate expression levels.
• Strong and tightly controlled promoters maximises protein production and reduces toxicity during growth phase
• However, very high transcription rates leads to an excessive metabolic load on the cell. This may decrease rate of product synthesis.
• To combat this, lower strength promoters may be used
• A range of promoter strength is necessary

Prokaryotic and Eukaryotic Promoters:

• Prokaryotic promoters contain 2 essential motifs (consensus regions) surrounded by spacer regions of RNA. These spacers RNAs are variable and can modulate expression levels
• Eukaryotic promoters have 2 (core and enhancer) elements -> different combinations of these can modulate transcription and modulate expression levels. Both core and enhancer elements have specific transcription factor binding sites (TFBS); these determine promoter function

Promoter engineering strategies:

1. Random mutagenesis using Ep-PCR (error-prone PCR)
   1. The TFBS is altered to make variant promoters with lower strengths.
   2. Application Example: Bacteriophage-derived PL- γ constitutive E. coli promoter: 196-fold range ⁶⁹ was engineered; each promoter had identical regulation. Optimal expression levels were dependent on strain background
2. Saturation mutagenesis of nucleotide spacer regions
   1. Only the variable spacer regions of the promoter are mutated; the consensus regions are retained
   2. Application Example: Lactococcus lactis promoter ⁷⁶ : 400-fold range in expression.
   3. Extending the idea: Mutations in the consensus region or alternating the length of the spacer region reduces promoter function dramatically, increasing dynamic range to 3 to 4 logarithms.
3. Hybrid Promoter Engineering
   1. Assembly of element-core promoter fusions to:
      1. Enhance basal core transcriptional capacity
      2. Enable novel promoter regulation
   2. Hybrid promoter core and enhancer elements are modular
   3. Hybrid promoters with tandem repeating UAS elements -> increase core promoter expression capacity
   4. Each additional UAS increases overall hybrid promoter strength
   5. Advantages include 1) generate large coverage promoter libraries 2) enhancing transcriptional capacity of strong endogenous promoters
   6. Application Example: Yarrowia lipolytica: the strong, highly regulated XPR2 promoter has a 105-bp upstream enhancer element, (UAS1B).96 Fusion of one - four tandem UAS1B copies to a core promoter created four hybrid promoters of increasing strength
4. Direct systematic modulations of TFBSs
   1. Application Example: e coli – 3 inputs from 4 different TFs modulated promoter strength over five decades

Paul J Rutten’s Thesis:

Aim(s):

Converting constitutive promoters -> (synthetic inducible promoters) SIPs; Characterise it using reliable predictive model.

Introduction:

• Very strong constitutive promoters means that SIPs created from it have a poor ON:OFF ratio, i.e. very little difference in the uninduced state and induced state.
• 2 types of TFs and operators
  • Activating operators – turns on the inducible promoter, which normally has weak basal activity
  • Repressing operators – turns off the inducible promoter, which normally has strong basal activity
• Optimal placement of the operator sequence in SIP depends on the TF
  • Repressing operators are most effective in the core region >proximal > distal
  • Activating operators are most effective in the distal region (only)

Conclusion:

• Libraries of new SIPs controlled by the XylR, LuxR and AraC TF were constructed from the Anderson set of 8 constitutive promoters (3 )
• The WANG AND-gate was replicated with these SIPs
• Promoters were weaker in the SVd context, showcasing context-sensitivity
• The relative promoter unit was a good comparator of promoter strengths across different contexts
• AraC constructs failed as SIPs were unable to be made from them.

Future directions:

• Build and characterise more SIPs from untested constitutive promoters to clarify the range of promoters that can be used to construct SIPs.
• Find the extent to which SIPs are context dependent.
• Use SIPs for forward engineering