We decided to use two different kinds of promoter systems. As a constitutiv pomoter, we chose J23100 from the registry. This promoter is placed on a plasmid called J61002. We had to place our operon behind the promoter onto J61002. So we cut J23100-J61002 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 (J23100-K1497007-J61002) were purified after incubating for 16 h at 37°C. But we still had our insert on a AMP vector. So we cut J23100-K1497007-J61002 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.