Part:BBa_K4144041

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 three 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.3).

Figure. 3 Construction of the selection plasmid

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Result

Using MEGAWHOP, we introduced site-saturation mutagenesis into three different residues, I79, F161 and L296, which locate in the binding pocket of IPTG [1]. After DpnI digestion (Fig.4), the mutated plasmids are transformed into E. coli BL21(DE3). The protein selection is carried out by a series of selective culture, using characters of our reporter genes integrated into the plasmid. Considering the inaccessibility of lactose due to its natural metabolism in bacteria, we applied IPTG as an alternative to maintain the stable induction during the process.

Figure. 4 Mutagenesis library construction.
A: No megaprimer in MEGAWHOP; B: Megaprimer added in MEGAWHOP. DpnI digestion is applied to both group, and non-mutated plasmid can’t be transformed into competent cells.

Our first attempt using sacB had failed, so we instead utilized KanR to bring out comparison experiments to compare the behavior of variants under different concentrations of IPTG. Gradient of IPTG including 0, 0.1, 0.5 and 1mM were added into 50ud/ml kanamycin plate. In total more than 30 mutants were identified and various kinds of behaviors occurred (Fig.5).

Figure. 5 Mutagenesis library construction. A: No megaprimer in MEGAWHOP; B: Megaprimer added in MEGAWHOP. DpnI digestion is applied to both group, and non-mutated plasmid can’t be transformed into competent cells.

In order to evaluate the behaviors of different mutants for better judgement, we designed an algorithm to access the IPTG responsive ability of the strains. We used number 1-7 to measure the intensity of the colonies on each plate, then integrated them into a heatmap for visualization (Fig 3.5). Our goal is to select a LacI variant that cannot sense the low concentration IPTG and at the same time normally functions at with concentrated IPTG induction. Thus, reflecting on different colors, the ideal strain is supposed to have lighter color between darker ones at high range of IPTG. Comparing the data, Strain #29 is exactly the one that we are looking for (Fig 3.4 C). Meanwhile, a few growths under 0.8mM IPTG guarantees that the mutant LacI still keeps the ability to release from promoter and it could function normally in our oscillatior - “Repressilator”. Selected mutant also appeared to solve the leakage problem that original LacI contain, showing a more outstanding performance.

we successfully derived the mutant that only respond to high concentration of IPTG (0.5mM), no growth is witnessed at lower concentration IPTG (Fig.3). This evolution also greatly solves the leakage problem of original LacI protein, as no colony grows without IPTG induction.