Materials and Methods
Forward and reverse primers were designed for RFP mRNA amplification:
- forward primer:
- reverse primer:
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 (Ct). The amount of RNA present in the sample can be calculated as follows: if sample A showed a Ct of 8 and sample B showed a Ct 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.
Results of fluorescence spectroscopyInvestigating the grown cell cultures by fluorescence spectroscopy clearly showed emission at 590nm (excitation at 540 nm) indicating a successful expression of RFP.
|Promoter||Emission at 590 nm|
192 +- 13
30 +- 1
345 +- 4
Results of RT-qPCR
Figure 1 shows the fluorescence signal over cycle number for amplification of the target cDNA of E. coli cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). As expected, the E. coli without the plasmid (-) showed amplification of unspecific targets (figure 2), which can be derived from high Ct values. This indicates, that the primers we designed bind to RFP cDNA specifically.
To quantify the expression levels of RFP under different promoters, the cDNA of a house-keeping gene was amplified as well. After we spoke to Dr. Trachtmann, who is an expert in performing and evaluating RT-qPCR experiments, we chose a GTPase-activation protein (GAP) as a housekeeping gene. The specific primers to amplify the desired house-keeping gene were kindly provided by Dr. Trachtmann.
Figure 3 and 4 showed that GAP is present in all samples. Under the assumption, that GAP is expressed under constant rates, one is able to quantify the expression of the target gene relatively to the expression of the house-keeping gene. Analytik Jena provided a qPCR device, which came with a software to interpret our results using the ddCt-method. Using this method, revealed the results shown in figure 5.
Figure 5 - Relative quantification of RFP expression compared to the expression of the house-keeping gene GAP in E. coli MG1655. Cells contained the plasmid BBa_J61002 with different promoters: BBa_J23100, BBa_J23112 and BBa_J23118. As a control E. coli MG1655 were cultivated without the plasmid.
RFP expression was about 7-fold higher than the expression of GAP in cells containing promoter BBa_J23100. Compared to that, the expression of RFP in cells containing promoter BBa_J23112 was about 55-fold higher than expression of the house-keeping gene. The highest expression of RFP compared to GAP was observed in cells containing promoter BBa_J23118. Here, RFP expression was 74-fold compared to GAP expression.
The melting curves showed that specific amplification products were generated (figure 6, figure 7, figure 8). As E. Coli MG1655 does not contain any target sequences for RFP primers when no plasmid is present, the melting curves show unspecific product generation (figure 9).
Results of agarose gel electrophoresis
Exposing the gel under UV light, reveals that the expected amplification product was generated (about 350 bp).Figure 10: Amplification mix loaded onto a 2 % agarose gel reveals production of one amplification product for cells containing the plasmid with different promoters.
We were surprised by the results of the characterization of the Anderson promoters as they do not meet our expectations. Actually, we expected that the use of different promoter strengths would result in different levels of mRNA in a way that weak promoters would result in weak fluorescence and low corresponding mRNA levels and strong promoters would lead to high corresponding mRNA levels. More precisely, we expected cells with the promoter BBa_J23100 to exhibit the highest mRNA level, as it was supposed to be the strongest promoter of the ones we characterized.
When measuring fluorescence, we obtained the expected results: it was revealed, that cells with the promoter BBa_J23100 (strongest promoter) produced more RFP than cells with the promoter BBa_J23118. Cells with the weakest promoter (BBa_J23112) produced fewest amounts of RFP.
Possible reasons for the discrepancy between promoter strength, RFP amount and mRNA concentration could lie in the promoter library itself: Changes in the promoter sequence to generate the library could lead to impaired secondary structure of the mRNA. This in turn may influence the binding of ribosomes and the translation initiation efficiency significantly.
It may be possible, that the differences in fluorescence are mainly caused by changes on the translational level and not on transcriptional level, as to date promotor strength for these biobricks was only determined by fluorescence measurements.