Remastering Phagemid Vectors - Engineering Assignment Help

Assignment Task

Abstract -
Well characterised transcription factors and their DNA operator binding sites are used in synthetic biology to design genetic circuits.
These are widely used for biomedical and biotechnological applications, performing user-defined functions to output predictable gene expression patterns.
For the field to expand, new transcription factors must be identified or engineered by evolving old ones to recognise new DNA operator binding sites. Brodel et al. (2016) engineered 12 new cI? transcription factors. However, the phagemid vectors that carried them imposed high metabolic burden on host cells. This work 'remastered' these 12 phagemids from high to low burden, through introducing two-point mutations. A significant reduction in metabolic burden was confirmed through E. coli growth culture assays. In addition, it was shown that the mutations did not disrupt cI? transcription factor activity, or the phagemid’s capacity to function within the phagemid-assisted directed evolution system. Hence, these new low-burden transcription factor phagemid vectors are now suitable for downstream synthetic gene circuit engineering and for deposition into the Addgene public repository. The study highlights the importance of understanding metabolic burden in the synthetic biology field.

Introduction -
Transcription factors (TFs) and their DNA operator binding sites are used within synthetic gene circuits. They are arranged in such a way to carry out user-defined functions to output predictable gene expression patterns, and are becoming increasingly useful for biomedical and biotechnological applications (reference). The first dynamical synthetic circuits were built using just a few TFs and their operators, such as the genetic toggle switch (gardner et sl 2000). Though the field is growing, increasing circuit complexity is limited due to the small number of TFs available in the synthetic biology toolbox (brodel et al 2016,Brophy et al 2014, gardner et al 2000, elowirz and leibler 2000,Guet et al 2002, becsei and serrano 2000). Expanding this toolkit through engineering TFs with different properties and orthogonal activities is therefore a topic of current focus (Brodel et eal 2016, milo et al 2002).

Brodel et al. (2016) addressed this subject by building a protein directed evolution or ‘evolver’ system containing multiple gene circuits. Their phagemid-assisted continuous evolution (PACEmid) system utilises the M13 bacteriophage life cycle to evolve new, orthogonal TF-operator pairs (PACE references, brodel et al 2016). The system involves three plasmids, one of which is a packaged phagemid, the focus of this study, containing a the TF gene to be evolved. As an essential phage gene is under conditional control - it is only expressed when the TF binds a new operator sequence – PACEmid selects for the new TF-operator pair over time (Brodel et al 2016).
Brodel et al. (2016) used PACEmid to evolve 12 new variants of the bacteriophage ? repressor TF, encoded be gene cI (lee et al 2007, brodel et al 2016,lutz et al 1997, ptashne 2011). ? repressor harbours dual activator/repressor activity, and competes with TF Cro at a natural genetic toggle switch in bacteriophage ?’s genome (gardner et al 2000, ptashne 2011). This switch consists of a bidirectional promoter (PR/PRM) containing three ? repressor operator binding sites. Six new PR/PRM were designed and constructed with different operator sequences for ? repressor to evolve to bind to (brodel et al 2016). Brodel et al. (2016) derived 12 new, completely orthogonal cI? variants that bind these new operator sequences: six evolved from the cI wildtype backbone, and six from a cIopt backbone (with stronger PRM RNA polymerase recruitment activity) to enable each two different variant activating strengths (brodel et al 2016).

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