Forward and reverse primers were designed for RFP mRNA amplification:

• forward primer: atggcttcctccgaagacg
• reverse primer: actcaccgtcttgcaggg

The expected amplification product has a length of 352 bp.

Lyophilized biobricks were resuspended in 10 µL ddH₂O according to the provided protocol. A vial of competent cells was retrieved from storage at -70 °C and thawed on ice. Then, 1 µL plasmid DNA was added to 80 µL electrocompetent E. coli MG1655 and gently mixed. The cell-DNA suspension was transferred to a chilled electroporation cuvette. Electroporation was performed at 2500 V. Cells were immediately recovered in 1 mL pre-warmed 2YT medium and incubated at 37 °C for 1 h. Aliquots of the recovery medium were plated out on agar plates containing 100 µg/mL ampicillin. The plates were incubated overnight at 37 °C. Single colonies were picked from the plates and grown overnight in 5 mL 2YT medium supplemented with 100 µg/mL ampicillin in glass test tubes at 37 °C and 130 rpm. The optical density (OD₆₀₀) of the overnight cultures was determined and a 5 mL main culture in 2YT medium supplemented with 100 µg/mL ampicillin in glass test tubes was inoculated with a starting OD₆₀₀ of 0.2. After 3 h of growth fluorescence of 50 µL of the cell suspension was measured (excitation: 540 nm and emission: 590 nm). Also, 500 µL of culture was centrifuged and the pellet was used for RNA extraction. This was done using the Zymo QuickRNA Fungal/Bacterial Mini Prep Kit following the user manual. In addition to that, E. coli MG1655 was cultured and harvested for RNA isolation without the plasmid BBa_J61002 to ensure, that our primers specifically bind to RFP mRNA.

Subsequently RT-qPCR was performed. For the reverse transcription 0.5 µL random hexamers (stock = 50 µM), 0.5 µL dNTPs (stock = 10 mM each), 0.5 µL RNA template and 5.5 µL RNAse-free water where mixed and incubated at 65 °C for 5 min, followed by 5 min on ice. The reverse transcription mix was prepared and added to each reaction. It composed of 2 µL 5x SSIV buffer, 0.5 µL DTT and 0.5 µL SSIV per reaction. The mixture was incubated at RT for 10 min, followed by incubation at 50 °C for another 10 min. The reaction was inactivated by incubation at 80 °C for 10 min. QPCR was performed in triplicates using KAPA SYBR FAST qPCR Mix (2x). 10 µL 2x KAPA Mix, 0.4 µL each forward and reverse primer (stock = 10 pmol/µL), 1 µL cDNA and 8.2 µL water were mixed. Initial denaturation was performed at 95 °C for 3 min, followed by 44 cycles of denaturation (95 °C, 15 sec), annealing (58 °C, 30 sec) and elongation (68 °C, 45 sec).

SYBR, included in the KAPA mix, binds to double stranded DNA absorbing blue light and emitting green light. Therefore, the fluorescence signal increases with the increasing amount of double stranded DNA. The higher the concentration of the template in the sample, the faster the fluorescence exceeds the threshold value. The number of cycles at which this happens is called threshold cycle (C_t). The amount of RNA present in the sample can be calculated as follows: if sample A showed a C_t of 8 and sample B showed a C_t of 11, sample A contained 2³ = 8 times more template.

A melting curve was run afterwards in a temperature range between 60 °C and 93.9 °C (in 1 °C steps), to verify, that one specific amplification product was generated.

To validate, that the expected amplification product was generated, 5 µL of the amplification mix was added to 1 µL of 6 x loading dye and loaded onto a 2 % agarose gel. A marker was also loaded (ThermoFisher 1 kb Plus DNA ladder). In addition to that, the gel contained the nucleic acid stain GelRed to visualize the nucleic acid when exposed under UV light. The gel was run at 80 V (1V/cm) for 1 h.