Difference between revisions of "Part:BBa K4144041"

Revision as of 20:49, 11 October 2022

Impreoved LacI protein responsive to high-level lactose only

Background and purpose

In order to regulate the SAMe production in a periodic manner, we designed Oscillator Module using a classic oscillator "the Repressilator". The oscillator contains three repressors, one among which is the LacI (Fig.1). However, since lactose is commonly existed in daily diet and thus intestinal environment, the oscillation would be easily interrupted. Driven by the need in the Oscillator Module of our project, we set up Directed Evolution Module to develop the LacI repressor. Our goal is to derive the LacI variant that couldn't sense low concentration of lactose and only respond to high-level lactose which naturally wouldn't appear in gut environment.

Figure. 1 Diagram for oscillator "the Repressilator"(Elowitz, M., Leibler, 2000)

 The basic principle of directed evolution is shown below (Fig. 2): building rounds of mutagenesis, selection for ideal members and amplification, that is, generating a library of variants, separating target desired members by setting multiple conditions and then enlarging them as the templates of the next round.

Figure. 2 Principle of directed evolution cycle

Design of the selection

 In order to separate desired LacI, we constructed an expression element where two reporter proteins are placed downstream of LacI promoter. The first one is SacB protein (BBa_K22921). This part encodes the Bacillus subtilis levansucrase which catalyzes the hydrolysis of sucrose and synthesis of levans that is lethal to gram-negative bacteria such as E. coli. The second one is KanR, which provides the resistance to kanamycin. We also included a reporter mRFP1 by using BBa_J04450 as vector backbone of our plasmid, of which fluorescence could aid in the judgement. In addition to these, a LacI controlled by a constructive promotor (BBa_J23116) is also included. The schematic diagram is showed below (Fig.2).

Sequence and Features