As the second promoter system we chose an inducible one. Therefore we took a T7 promotor on a pSB1AK3 vector from the registry. We had to place our operon behind the promoter onto pSB1AK3. So we cut T7-pSB1AK3 with SpeI + PstI and dephosphorylated the digestion. Furthermore we digested B0034-4CL-B0034-TAL-B0034-CHS-B0034-CHI-pSB1C3 (K1497007) with XbaI + PstI. We ligated both digestions with T4 ligase and transformed the products into Top10 E.coli via heat shock. We spread out on AMP-LB agar plates and incubated the plates overnight at 37°C. We analyzed obtained colonies by colony PCR and agarose gel electrophoresis.

Positive colonies were inoculated in 5 ml LB with AMP and plasmids (T7-K1497007-pSB1AK3) were purified after incubating for 16 h at 37°C. But we still had our insert on a AMP vector. So we cut T7-K1497007-pSB1AK3 with EcoRI + PstI and ligated this part with a previously digestion of mRFP-pSB1C3 with EcoRI +PstI. We transformed the ligation in Top10 via heat shock and spread out on CMP-LB agar plates. After incubating the plates overnight at 37°C, we analyzed white colonies by colony-PCR and agarose gel electrophoresis. Positive colonies were inoculated in 5 ml LB with CMP and plasmids were purified after incubating for 16 h at 37°C.

For further verification we send the samples to Eurofins for sequencing.