Stanford iGEM Team Wiki

Novel Directed Evolution Selection Schema



Like PACE, PREDCEL+ relies on M13 bacteriophage to carry a library of mutant genes. The replication of these viruses is tied to the fitness of the mutant gene such that the most fit gene mutants produce more offspring, which eventually become the majority of the viral population. Conventional PACE and PREDCEL creates this linkage by creating a biocircuit whose output is M13 gene III (gIII), which encodes a phage protein (pIII) involved with phage entry into the cell. However, even low amounts of gIII expression render cells resistant to phage infection, complicating matters. We decided to use a different phage protein involved in mediating phage entry, pVI (encoded by gVI), for our selection based on findings that it does not render cells resistance to further infection, is unstable in the absence of pIII, and leads to higher phage production when expressed at higher rates.

One roadblock encountered early-on when using PACE was that phage may not be able to propagate in culture if they all encode mutants poor at performing the task to be evolved. This leads to ‘phage washout,’ where evolution halts due to the inability of the phage to replicate. This issue was solved by using a genetic element termed a ‘drift cassette’, which can inducibly express gIII or gVI when needed. Typically, drift is induced earlier in the evolution and reduced gradually to zero as the evolution progresses. However, it is unclear how much or when drift should be induced without periodically conducting plaque assays to visualize the amount of phage at any given moment. The Stanford 2019 iGEM team thus developed an ‘infection reporter’ based on GFP fluorescence output to report on what fraction of cells at any given moment in time are infected with phage. GFP expression is driven by the phage shock promoter (Ppsp), which responds to filamentous phage infection (specifically, expression of M13 gIV, which causes pores in the E. coli membrane that allow phage progeny to leave the cell) in addition to osmotic shock, organic solvents, and blockage of secA machinery used for protein export. We used two sequences for Ppsp in our designs - one described by the 2018 FSU iGEM team (part BBa_K2832003) and the other the wild-type Ppsp in plasmid pDB023f1 from Prof. David Liu’s lab (part BBa_K3258000).

To further simplify the cloning of PREDCEL+ and limit the possibility of phages containing mutant proteins propagating outside of engineered host cells, we split our system into three plasmids, which we term the helper plasmid (HP), accessory and mutagenesis plasmid (APMP), and the selection plasmid (SP). The components of each are detailed in the figure below:

Figure 1.

To test our designed PREDCEL+ architecture, we decided to recapitulate the evolution done on the T7 RNA Polymerase to allow it to recognize the T3 RNA polymerase promoter conducted in the seminal PACE paper1. We thus used a more specific APMP and SP. All plasmids used are outlined below:

Figure 2.

Novel Selection Schema Using PREDCEL+

Once validated, we aimed to use PREDCEL+ to test out a standardized selection schema for biosensor evolution. Making use of the discretized nature of PREDCEL, we thought it easy to transfer between multiple populations of bacteria in such a way that the gene to evolve is selected to be fit in all of them. We thought we could make use of this advantage to select for biosensors by negatively and positively selecting for and against binding, respectively, in the presence and absence of the desired molecule to sense13. As a proof of concept, we decided to evolve the lacI repressor to respond to new substrates and to adjust the dynamic range of the lacI IPTG dose response. To this end, we developed two APMPs - one for positive selection and the other for negative selection. All plasmids used for the evolution are presented below:

Figure 3.

Experiments and Results