At first, we tried to co-transform the pCold I-VSW-3 RNAPN-NpuN (<partinfo>BBa_ K4907148</partinfo>_pCold I), <i>L</i>-arabinose-induced SspC-VSW-3 RNAPC expression circuit (<partinfo>BBa_K4907116</partinfo>_pSB1C3) and the reporting circuit of the pVSW-3(18) promoter (<partinfo>BBa_K4907109</partinfo>_pSB3K3) into BL21(DE3). It should be noted that the <i>cspA</i> promoter on pCold I is an IPTG-inducible one because a copy of <i>lacO</i> is placed downstream the promoter sequence. Hence, in accordance with the ways of induction, the AND gate constructed here was a three-input one, linking the chemogenetics and thermogenetics (Fig. 13a, left). We set carefully the conditions of different control groups and measured the output signals after induction for 12 hours. As expected, when all the induction requirements were met (0.5 mM IPTG, 0.2% <i>L</i>-arabinose (m/v), cultivated at 25 °C), the normalized fluorescence intensity was significantly strongest (Fig. 13a, right). Besides, when one input was absent, the output signals were even about 4-fold lower than the “all-input-1” group and all these deficient groups exhibited an equal level of the weak output signals, which indicated that the three-input AND gate was very stringent.
