Team:BOKU-Vienna/Demonstrate
Demonstrate
Proof of Concept with 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.
To show that our engineered system and idea work, we performed quantitative measurements and achieved
qualitative results with experiments based on the color change of a sample, because of 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 (Part: 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_K592009. The expression of T7-Polymerase is followed by the transcription of the amilCP gene
as it can be seen in figure 1.
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.
All experiments were performed according to all rules and policies approved by the iGEM Safety
Committee. A summary of our safety regulations can be found under “Safe Lab Work” and more informations
are in our Safety Form.
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 Project Design or
Model
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 60 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 pages 44, 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:
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
Figure 5 shows the microtubes from our 6th experiment which can be compared with Table 8. The first sample is our negative control without any induction, followed by the microtube induced with 1 mM Theophylline after ½ hour of incubation. The third microtube is induced with 1mM Theophylline after 1 ½ hours of incubation, the 4th induced with 1mM Theophylline after 2 ½ hours of incubation and the last sample is induced with 1mM of Theophylline after 3 ½ hours of incubation.
Cell-free measurement
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 Terminator BBa_KB1001 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 induction of Mycolactone.
After 15h of incubation at 37°C on a shaker at 180rpm OD600 was measured and 1mL of the culture was
spinned-down, pellet washed with 1xPBS, spinned-down again and resuspended with 1mL 1xPBS. The
fluorescence of uninduced BBa_K3015002 was measured together 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. This equals around 66,000
fluorescence molecules per cell.
Unfortunately, the induction didn’t work in vivo. 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 50µL per well. The cell free samples (13 µl) were diluted to a total volume of 50 µl with
1xPBS buffer.
Testing in cell-free conditions showed that the Mycolactone aptamer riboswitch works, due to the produced GFP we observe and measured after induction with the toxin. Increasing plasmid and Mycolactone concentrations showed an evenly rise in GFP production (see figure 5).
Figure 6 presents the relative fold increase values resulting from rising Mycolactone concentrations, that were induced.
