Experimental Results of the Original Design

Verification of ΔcheZ strain (protocol)

    The size of colonies of E.coli strain RP1616 was much smaller than that E.coli steain RP437 under the same circumstance and after same period of incubation time(about 10h), and the result is shown in Figure1. In colony PCR using the primers of cheZ gene following , RP437 absolutely has much more outcomes than RP1616 and we conclude that RP1616 is actually a ΔcheZ strain.

Figure1.The result of colony PCR(From left to right, the first lane is the marker, the third and the forth lane is the PCR outcome of strain RP437 and strain RP1616, the sixth and the seventh lane is the PCR outcome of strain RP437 and strain RP1616.)

Verification of the function of the constructed riboswitch (protocol)

Figure2. The growing state of the reprogrammed bacteria with Aptamer-cheZ(Left:0.3%agar with 0mM Thephylline, Right:0.3%agar with 0.25mM Thephylline)

    From the results above we concluded that the constructed riboswitch functions well, especially on 0.3% Semisolid medium.

Verification of Toggle-switch (protocol)

Numbers of colonies with RFP: Numbers of colonies with GFP ≈ 8:25

Modulate Toggle-switches to produce more balanced ratio (protocol)

Numbers of colonies with RFP: Numbers of colonies with GFP ≈ 6:1

Test of Incorporated Riboswitch into one side of Toggle-switch (protocol)

Verification of the constructed system’s function (protocol)

• 6 tubes of constructed monoclonal RP1616

• Results of the sixth tube