Team:BOKU-Vienna/Results
Results
Overview
We designed a riboswitch-based readout that can be interpreted with the naked eye. The riboswitch consists of an aptamer-based expression of T7-Polymerase and indirectly amilCP under the control of the T7-Promoter. Our work is based on the idea of the specific binding of an inducer to an aptamer sequence.
We hereby proudly present you the following results:
- Quantitative measurement of the fold increase through GFP Fluorescence after induction with Theophylline
- Proof of concept with Part: BBa_K3015011- Qualitative measurement of our riboswitch expressing amilCP
- Successful screening of a Mycolactone responding aptamer
Quantitative measurements of the fold increase of GFP Fluorescence
In order to make the riboswitch screening easier, we wanted to provide comparable numbers about the leakiness of the uninduced module by testing the riboswitch with GFP as a reporter gene. By using the fluorescein standards from the measurement kit, fluorescence values can be converted into comparable units. Figure 1 shows the power function of the fluorescein standard curve (log scale). Net mean fluorescence values can be put into the power function as y-values to calculate the corresponding µmol/L fluorescence molecules.
For example: The Theophylline riboswitch with an uninduced net mean fluorescence of 1558.66 equals 0.07526 µM GFP-molecules. Through the OD600 value the number of cells per mL can be calculated. For Escherichia coli with an OD600 of 1.0 = 8*10^8 cells per mL. To measure reliable OD600 values, samples must be measured in the linear range of the photometer and therefore often need to be diluted and the real OD600 calculated. An OD600=3.81 the number of cells per mL equals 3,048,000,000. The concentration of fluorescent molecules divided by the number of cells per mL leads to 2.47 * 10^-20 mole per cell. Which are 15,000 molecules per cell. The activity and increase in gene expression after induction can be measured without standards. The net mean fluorescence values can be normalized to OD600=1.0 by dividing each fluorescence value through the corresponding OD600 value. The normalized fluorescent values can be divided through the uninduced fluorescence to obtain the relative expression or fold increase (see figure 2). Figure 2 shows the fold increase at different inducer concentrations. The concentrations of 0 µM, 1µM, 10 µM, 100 µM and 1 mM of Theophylline were used for induction. As presented in Figure 2, the Theophylline riboswitch shows low response to 1µM Theophylline induction, a 1.5 fold increase in the presence of 10µM Theophylline, a 4 fold increase of gene expression at 100µM Theophylline and a 6.4 fold increase at 1mM Theophylline.
The same measurement was done to characterize the GFP expression cassette BBa_J364001.
Around 93,400 fluorescent molecules per cell were produced at an OD600 of 3.75 after 15h of incubation.
Around 121,000 fluorescent molecules per cell were produced at an OD600 of 3.75 after 15h of incubation.
Proof of Concept with the composite Part BBa_K3015011
The ultimate goal of our new diagnostic method, called Mycolactone Diagnostics, was to achieve a clear naked-eye readout of samples induced with Mycolactone. The purpose is to make the diagnostic method as clear and simple as possible, for it to be employed without the need for observational instruments, trained professionals or specific knowledge.
In addition to quantitative measurements we wanted to achieve qualitative results with experiments based on the color change of a sample, based on the expression of a chromoprotein. For our construct we chose the part BBa_K592009 which is the Blue Chromoprotein amilCP due to the fact that it can be seen with the naked eye and doesn’t require any instruments to observe.
Moreover, another key aspect of our diagnostic method was the rapidity. Based on our modeling in which we predicted that the expression of Blue Chromoprotein should be visible 45 minutes after the induction, we measured the OD600 of our engineered E. coli DH10B to calculate the replication rate and visually analyzed the production of Blue Chromoprotein half hourly to hourly.
Another advantage of part BBa_K592009 is the codon optimization performed by Team Uppsala 2018. This allows the protein expression to be maintained for a longer period of time through several generations of growth, making the expression of the Blue Chromoprotein more stable.
Our experiments started with measurements crucial for an optimized, efficient and clear diagnostic process.
The measurements were performed with one of our constructs (BBa_K3015011) for a proof of concept.
The expression cassette consists of the following two constructs which are presented in figure 7 and 8. Figure 7 shows our T7-Polymerase gene under the control of a Theophylline inducible riboswitch (BBa_K3015004). The expression of T7-Polymerase is followed by the transcription of the amilCP gene as it can be seen in figure 8.
Since receiving the toxin Mycolactone was unsure until very late in our project and our possibilities in working with this toxin were limited because of its toxicity, the aptamer for our proof of concept is designed to specifically bind to Theophylline, a toxin we chose as an alternative to Mycolactone. This also shows the universal application of our idea of a toxin inducing a color reaction through binding to a specific aptamer.
First, we wanted to make sure that the binding of Theophylline to the aptamer sequence would induce a chain of reaction starting with a confirmation change by which the aptamer loses its hairpin structure. Therefore, intrinsic termination would not occur and the intrinsic
RNA-Polymerase is free to transcribe the RBS and gene of interest: the T7-Polymerase. The ribosome can bind to the ribosome binding site and translation of the T7-Polymerase takes place. In the next step, the produced T7-Polymerase binds to the T7-Promoter which results in the transcription of the mRNA for amilCP allowing the initial induction to be identified by a clear visual readout through the color change. For a more detailed description of all reactions please check out “Design” or “Modeling”
For proving the functionality of the switch, we set up overnight cultures with the plasmid construct induced with different Theophylline concentrations. Additionally, as negative control, samples with bacteria including our construct without the toxin were measured. After 15h of incubation time, following results could be seen:
From this experiment with induced overnight cultures we calculated the growth rate of the bacteria with the help of our OD600 results. We concentrated on the replication rate of our E. Coli DH10B strain, as well as the impact of different concentrations of Theophylline on the replication rate. Following calculations were made:
After performing this pre-experiment which gave us the confidence that our construct works, our constitutive promoter doesn’t leak and the T7 promoter works, we decided to concentrate on measuring the rapidity of the color expression process in our construct. Our theoretical model predicted the production of Blue Chromoprotein to be seen after 45 minutes. Therefore, we wanted to perform half hour- to hourly measurements of the construct. As can be seen from the calculations, our E. coli reached the stationary phase where expression of amilCP was rather low/slow, that’s why, for further experiments, we decided to induce bacteria with the toxin in their exponential growth phase.
Six experiments with the same set up and working protocol, which can be seen in our “Lab Journal” (on page 43, 46 and 47). were performed to assure the reproducibility of measurements.
For each experiment, with an overnight culture (15h of incubation time) of E.Coli DH10B with the construct plasmid a fresh culture was inoculated. This culture was incubated until reaching an OD600 of 0.6 to 1.2, however an OD600 of 0.8 to 0.9 proved itself to be most efficient for the quick color reaction. After reaching the optimal OD600 (0.8 -0.9.) in which the bacterial cells are in their exponential growth phase, test tubes filled with the overnight culture were induced with Theophylline in different concentrations, incubated and hourly analyzed. Following results were achieved:
1st experiment
2nd experiment
Figure 9 shows the samples from experiment 2 after being induced with different Theophylline concentrations and incubated for 1 hour (first row) and 2 hours (second row).
The first microtube in each row is our negative control without being induced with Theophylline, the second column each row is the culture induced with 0.001 mM Theophylline, the third microtube is induced with 0.01 mM, followed by 0.1 mM, 1 mM and 5 mM inducer concentration. (from low to high-comparable with the concentrations in table 4)
Figure 10 shows the samples from experiment 2 after being induced with different Theophylline concentrations and incubated for 3 hours (first row) and 4 hours (second row).
The first microtube in each row is our negative control without being induced with Theophylline. In both Figure 9 and 10 the color change can be visually seen as described in Table 4.
3rd experiment
4th experiment
5th experiment
6th experiment
Cell-free Measurements
The iGEM Team BOKU-Vienna created a Mycolactone riboswitch that works on a transcriptional level BBa_K3015000. We found the aptamer sequence in the paper: Samuel A. Sakyi, et al., October 2016. After choosing the most promising aptamer sequence (Aptamer 3683) and designing multiple proposals for intrinsic termination, we fused the aptamer sequence with intrinsic termination to a promotor and RBS to create the composite part Part:BBa_K3015001. It is important for the parts functionality to be placed between the Promoter and RBS, so the hairpin structure from the intrinsic termination can stop the RNA-polymerase before it can transcribe the RBS. To measure the leakiness GFP BBa_K3015013 as a reporter gene as well as the TerminatorBBa_B1001 were added downstream of the composite part BBa_K3015001. The resulting construct is composite BBa_K3015002.
The leakiness was investigated in vivo by measuring the GFP production without addition of Mycolactone. After 15h of incubation at 37°C on a shaker at 180rpm OD600 was measured and 1mL of the culture was spun-down, the pellet washed with 1xPBS, pelleted again and resuspended with 1mL 1xPBS. The fluorescence of uninduced BBa_K3015002 was measured, along with the fluorescein standard from the measurement kit (see figure 1) to convert the net mean fluorescence of the riboswitch into molecules per cell.
The arithmetic net mean fluorescence of 4733.22 from the uninduced BBa_K3015002 was put into the curve equation to calculate a concentration of 320 nM at OD600=3.65 (see Spreadsheet for raw data). This equals around 66,000 fluorescence molecules per cell.
Unfortunately, the induction by Mycolactone didn’t work in vivo (results not shown). Therefore, the riboswitch was tested cell-free with a myTXTL® Sigma 70 Master Mix Kit (sponsored by Arbor Biosciences). For Protocol see: Cell-Free Expression Handbook, June2019, page 10-13 (https://arborbiosci.com/mytxtl-manual/).
1µl Mycolactone dilution/Theophylline dilution/buffer respectively
+ 3 µL Plasmid dilution
+ 9 µl sigma 70 Master Mix
∑ 13 µl
The reactions were incubated in 1.5 ml reaction tubes for 6 h at 29 °C on a thermomixer.
Fluorescence was measured with the Tecan-Infinite-200-plate-reader. Black 96-well plates (flat bottom) were filled with 4 rows of standard (Fluorescein from distribution kit) in 1:2 dilution steps to a total volume of 50 µL per well. The cell free samples (13 µl) were diluted to a total volume of 50 µl with 1xPBS buffer.
Table 9: Fluorescence values from different plasmid concentrations with variating amount of induced Mycolactone
Testing in cell-free conditions showed that the Mycolactone aptamer riboswitch works, due to the produced GFP we observe after induction with the toxin. Increasing plasmid and Mycolactone concentrations showed an evenly rise in GFP production (see figure 13).
Figure 13 presents the relative fold change values resulting from increasing Mycolactone concentrations.
amilCP BBa_K592009 - Fluorescence
In addition to our successful qualitative measurements we also wanted to get some quantitative data regarding the expression of amilCP. Since the part BBa_K592009 . was not sufficiently characterized for detailed fluorescence measurements, the experiments were challenging. We measured the excitation maximum for Blue Chromoprotein at 369 nm and the emission maximum at 430 nm. With those parameters we conducted fluorescence measurements of our experiments. The positive outcome is, that with increased Theophylline concentration, the expressed Blue Chromoprotein is also increased, which can be seen in our qualitative results. Figure 14 presents the fluorescence of samples with different Theophylline concentrations as a function of incubation time. However, after 3 hours of incubation our numbers drop. A theory for this phenomenon is “Quenching”. We assume that the decrease in fluorescence intensity can be affiliated with excited state reactions, energy transfer, complex- formation and collisional quenching. For more information please check out https://www.sciencedirect.com/topics/engineering/quenching.
Furthermore, we also measured the absorbance intensity at the given maximum at 588 nm.
To our surprise, the numbers for our white/yellow negative sample, which was not induced with Theophylline were much higher than our positive samples, which can be seen in
Figure 15. This could either mean that the given absorbance maximum at 588 nm is not correct for measuring amilCP or that the measurements should not be performed in vivo.
For further experiments and measurements of the expression of amilCP we would first want to characterize the protein for correct fluorescence measurements and more important work with a standard. In addition to that the extraction of amilCP from the cells should be considered for clearer measurements.
Theophylline Aptamer BBa_K3015007
The Theophylline aptamer with intrinsic termination worked in multiple experiments. Figure 16 shows the increase in gene expression after induction. The riboswitch is increasing gene expression by 400% in presence of 100 µM Theophylline and 640% in the presence of 1mM Theophylline in vivo. You can find more information on the registry page BBa_K3015007
BBa_K584000
When designing our project, we planned on incorporating a viability signal that expresses GFP in case of living cells under the control of an arabinose inducible promoter (pBAD). Since the construct that we cloned contained an arabinose inducible promoter (BBa_I13453 ) that didn’t respond to induction despite correct sequence, we decided to test the respective composite part BBa_K584000 (pBAD + GFP generator) under different arabinose concentrations.
As can be seen in figure 17: The arabinose inducible promoter didn’t respond. To verify this data, we repeated the experiment, with the same result (see figure 18).
As can be seen in figure 17 and 18: The promoter didn’t increase GFP expression in a significant way. The fluorescence of 0.04 µM Fluorescein is comparable to a medium strength promoter at an OD600 = 0.5.
After doing some research we figured out that the strain we used (Escherichia coli DH10B) most likely lacks crucial genes that transport arabinose into the cell or is expressing genes for arabinose degradation. If we were to go forward with this project, we would either choose a different strain (e.g. DH5alpha) to work with or try to incorporate transporter genes.
