Team:Bielefeld-CeBiTec/Engineering

Bielefeld-Cebitec-2020-Engineering

-->
Engineering

Part 1 – The PCB

If we think about engineering, it is all about imagining a new system, design, build and test our approach, realize improvement potential and start again with redesign. This is called the design-build-test cycle – The fundamental concept of engineering. One example of how this concept is applied in our project, is the construction of our Printed Circuit Board (PCB). The PCB is one of the most important parts of the technical realization. But let’s start with the beginning - the design. From there our design, the build test cycle starts.

Design

Fig. 1 - 3D Model of the PCB

For the design of our PCB we worked together with the CITEC of the University of Bielefeld to define the technical implementation of our project idea and discussed the practical realization. The design of the board, as well as the schematics, were created by the working group "Cognitronics and Sensor Systems" according to our requieremnts to use the PCB for SAW masurements. To be able to measure a phase shift at all, it is possible to design various circuits. Therefore, we discussed two possible realizations of the PCB. One solution was to use an analog mixer. An exclusion criterion is the amplitude dependence of an analog mixer. This is problematic due to the fact that at the time the circuit diagrams were developed, we did not have any knowledge of the amplitude of the frequency passing through the SAW chip. Therefore, we decided to search for another approach. A second possibility, which was finally chosen, was the use of an XOR-gate. The functionality of the XOR is described in the Project/Hardware section. We attempted to capture the signal curve of the XOR at the contact points to visualize it on the scope. The signal curves of the XOR are shown in Fig. 2 and 3.

Fig. 2 - XOR curve on the scope.

Fig. 3 - XOR curve on the scope.

Build

With the big support of the CITEC, we were able to realize our idea and build our own PCB. Since there was no sensing chip, no real phase shift could be evaluated by the antibody binding. So we had to simulate artificial phase shifts via the pin headers directly on the PCB. The areas marked in red in Fig. 4 can be connected in bridges, allowing a total of 16 different time shifts (in ps) to be simulated.

Fig. 4 - PCB Pinheader Marked.

Test

In order to cover the entire bandwidth of the DDS, we have generated the frequencies from 50 MHz to 600 MHz in 50 MHz steps for all 16 time shifts. We were able to detect the analog to digital converted voltage values on the serial console of the Arduino IDE. The results can be found in a table available for download below and in figure 5 below.

Download Table

Fig. 5 - Matlab Plot of the derived phase shift and time shift in 2D and 3D illustration.

The 3D plots illustrate the dependence of the time shift in [ps] or the phase shift in [°] and the frequency in [Hz] to the resulting voltages in [V]. At first, it must be mentioned that the time shift was initiated with a specified track extension for a signal, which might cause the procedure to be incorrect. Furthermore, the phase shift is derived based on the time shift, which can also be a source of mistakes. We can prove by our measurement that our PCB delivers a voltage change at the output in case of a phase shift on the input, which we could detect by reading the analog output. However, it must be clarified that our PCB model is not able to detect a perfectly valid phase shift as well as voltage change.

Improve

To measure the precision of our board, further tests have to follow. The maximum mistake must be calculated to receive the percentage deviation. Moreover, the DDS generates the frequencies with a certain interference factor as well, which has already been analyzed in the section "Frequency spectra of the DDS". Looking again at Figure 5, it becomes clear that e.g. there is supposed to be a minimum with a phase shift of 180°. But this point is located at a phase shift of 150°, which results a deviation of approximately 30°.

In conclusion, it can be said that even though there have been a number of systematic measurement deviations, we have developed a functional evaluation board that allows us to determine a phase shift from the analog output voltage value.

Files to download

To allow this project to be executed by further iGEM teams and possibly expanded, we have uploaded the files for the design of the PCB into our github repository. These files are available to download for free. Furthermore, one can find the source code for the Arduino Mega, which is used to read the PCBs analog output and additionally simulate phase shifts. We also uploaded the table of the results of the measurement as well as the matlab-plot so that the 3D plots can be reconstructed and viewed in three-dimensional. All these files can be used by future iGEM teams to do their own design, build and test cycles to use and improve our PCB prototype for their projects.

Part 2 – Engineering of FbFPs

Fluorescent proteins are a fundamental building block in synthetic biology research. Therefore, we decided to contribute an improved version of flavin-based fluorescent proteins (FbFPs) to the iGEM community. Due to their small size and their functionality in oxygen-free environment there are a perfect alternative to the commonly used fluorescent protein GFP.

Design


After a long research for a FbFP candidate, we found the protein SB2 from Pseudomonas putida, which shows a measurable fluorescent activity. So, we designed a testing system to measure the fluorescent activity of the SB2 protein (BBa_3410007) in contrast to the FbFP protein available in the parts registry of iGEM (BBa_K3013002). For this purpose, we chose the vector pJoe as our backbone and cloned the testing construct in silico with SnapGene (figure 6).

Figure 6: Plasmid map of the vector pJoe with the insert SB2

Build

With the in silico design plan, we were able to start with the construction of our vector. First, we decided to made a gradient PCR to identify the ideal temperature for the primer annealing. We measured the best annealing temperature at 63,2°C (figure 7).

Figure 7: Gel image for control of the PCR of the SB2 protein, the registry part and the vector pJoe.

With Gibson Assembly we were able to clone the amplified inserts into the pJoe backbone and transformed them in E. coli DH5α. Afterwards, we made a colony PCR to determine the clones carrying the right insert (figure 8) and validated the results with Sanger sequencing.

Figure 8: Colony PCR from SB2, the registry part and protein KOFP-7 in three different gels

The results of both steps prove the successful cloning of the inserts inside the pJoe vector. Finally, to measure the fluorescence, a retransformation from the strain DH5alpha to BL21 (D3) was performed, since pJoe carries a T7 promoter and requires a strain carrying a T7 polymerase for expression.

Test

For the characterization, the liquid cultures were induced at an OD(600)=0.7 with 0.1mM IPTG and then incubated for 16 hours at 20°C. After 10 hours the protein expression was measured. For this purpose, the emission was recorded via plate reader at an excitation of 450nm and an emission of 495nm. To measure the fluorescence, the cells had to be washed with PBS buffer beforehand, because the LB medium falsified the measurement. Therefore, the samples were centrifuged, decanted and resuspended in the PBS buffer. The fluorescence of the cells in PBS was then measured in the plate reader (figure 9).

Figure 9: Results of fluorescence measurement after 10 to 16 hours after induction with IPTG in the Tecan reader

Figure 9 shows a slightly higher fluorescence for the cloned protein SB2 (Original, BBa_3410007) in contrast to the FbFP from the part registry (Parts, BBa_K3013002). Therefore, we were able to design, build and test an improved FbFP for the iGEM community.

Improve

The results in figure 9 show a slightly higher fluorescence for our designed part. More research on this protein SB2 could consult in potential point mutations to grant a higher stability or a stronger fluorescence signal, which would improve the fluorescence even more. Future iGEM teams could use this part to design better FbFPs to establish this interesting class of fluorescent proteins deeper in iGEM.