We used SaeRS, a two-component system(TCS), to detect the concentration change of Zn2+. Then, we selected the fluorescent protein mCerulean as the downstream gene that can produce a fluorescent signal to showcase the activation of SaeRS.
Zn2+ can bind with SaeS protein, a kind of histidine kinase that can activate response regulator SaeR, then the mCerulean protein will be produced.
Under the concepts of SaeRS and mCerulean, we designed two composite parts, BBa_K4822005 and BBa_K4822007, representing the upstream and downstream components of the TCS signaling pathway, respectively. For more information about our parts, please refer to the Parts page.
We used three methods to ensure the success of the engineering:
To effectively assess the functions of BBa_K4822005 and BBa_K4822007, we combined them to create a complete signaling pathway plasmid named pSB1C3-phla-mCerulean-p3-SaeRS plasmid.
After retrieving the plasmid of Phla promoter, SaeRS and mCearulean, we amplified the sequence of these DNA fragments through Polymerase Chain Reaction. In the beginning, we directly used the typical PCR programs for each fragment, finding that the amplification results were unsuccessful after several tries. Therefore, we conducted gradient PCR to discover the favorable temperature conditions.
After the success of PCR, we used Gibson DNA assembly method to combine the above-mentioned DNA fragments. Then, we transformed the plasmid into E. coli through heat shock, checking the success of Gibson assembly through enzyme digestion after plasmid extraction from the engineered bacteria.
We picked several colonies of transformed bacteria and amplified them in liquid LB + CM media to perform plasmid extraction. From Figure 2, we see that colony 16 had the desired complete plasmid.
Next, we extracted protein from the successfully transformed bacteria and performed SDS-PAGE. We only performed protein purification and SDS-PAGE on the SaeS protein since confirming the expression of one protein is to ensure the expression of the gene saeRS. Figure 3 shows that the bacteria expressed the saeRS gene and produced the protein.
Last but not least, after the measurement of SaeRS function, we notice the noticeable correlation between fluorescent density and Zn2+ concentration.
To obtain further details about the experiments and their outcomes, please refer to the Experiments page and the Results page.
In the SaeRS functional assay, we excluded data with OD600 measurements below 0.2 to mitigate significant errors caused by the limited bacterial numbers. We speculate that the growth issues observed in groups with high Zn2+ concentrations are likely due to elevated osmotic pressure. Therefore, dealing with the osmotic pressure problem would be our future steps for improvement.
Furthermore, we observed that a noticeable correlation between fluorescent density and Zn2+ concentration was only evident within the range of 1-1.4 mM of Zn2+ concentration. To expand the range over which a noticeable correlation is observed for Zn2+ concentrations, it may be possible to achieve this by reducing the testing gap between Zn2+ concentrations and constructing the plasmid with a stronger promoter in the future steps.
Building on the methods mentioned above, although there were some design shortcomings that could be improved, we have demonstrated the initial success of our plasmid construction, which involves saeRS and the fluorescent protein gene. This accomplishment marks a significant milestone for the upcoming phases of plasmid design.
We co-transformed mtrCAB and ccm plasmids into E. coli. MtrA, MtrB, and MtrC are the major c-type cytochromes involved in EET, and ccmA-H is necessary for cytochrome maturation.
To connect with the upstream genes, we replaced the mCerulean part of the plasmid constructed in cycle 1 with the mtrCAB to construct a “saeRS+mtrCAB” plasmid. Then, to support cytochrome maturation, we construct another plasmid containing ccmA-H genes.
In addition, we made use of CoverWell Perfusion Chamber to build up a simple device to check the functionality and feasibility of the engineered E. coli.
We used several methods to ensure the success of the engineering:
We amplified the MtrCAB fragment by adding two restriction enzyme cutting sites, SacII and BamHI, at both ends through PCR and used enzyme digestion to obtain the mtrCAB and saeRS DNA fragments. After digestion, we ligated them together and completed saeRS+mtrCAB plasmid construction.
Next, we extracted and used the PCR technique to amplify the ccmA-H part of the gDNA of E.coli str. K-12 substrain MG1655. Then, we used enzyme digestion and ligation to combine ccmA-H genes with replication origin from pYJ-phla-LacZ, BBa_J23100 and BBa_B0015 terminator from pSB1C3-mCerulean.
After saeRS+mtrCAB plasmid and ccmA-H plasmid were successfully constructed, we co-transformed them into E. coli strain BL21 to obtain expression strain.
After we ligated the mtrCAB and saeRS DNA fragments together, we transformed into DH5α and inoculated on LB+CM plates in 37°C incubator overnight. The next day, we didn’t see any colonies on the plates. It seemed that our transformation failed.
For ccmA-H plasmid, we also ligated the fragments together, transformed the plasmid into DH5α, and inoculated it on LB+Amp plates in 37°C incubator overnight. The next day, we could see the colonies on the plates and then inoculate them into 5 ml LB+Amp liquid in 37°C shaking incubator overnight. We extracted the plasmid and digested it by restriction enzyme to check if it was the correct plasmid. However, the DNA band was not at the correct position after electrophoresis.
For saeRS+mtrCAB plasmid, there were some reasons resulting in the failure. For example, we speculated that the fragments were not ligated successfully, or the plasmid was not transformed into DH5α. After we retried the plasmid construction carefully, we finally constructed saeRS+mtrCAB plasmid. For more details, see the Wet Lab Results.
However, we failed to construct ccmA-H plasmid. We speculated that something wrong happened during ccmA-H PCR. We would construct ccmA-H plasmid from PCR again. Do it carefully to see where it went wrong.
In the initial stages of our device design, we considered several key concepts mentioned below.First and foremost, we would like to build a portable device that works as a fast test kit. In addition, our other primary objective was to create a design that effectively shields the device from external ambient light, ensuring the utmost precision in our measurements of fluorescent proteins. Furthermore, we aimed to equip the light source with the capability to emit light across various wavelength ranges, tailored for the excitation of fluorescent proteins. This approach offers a dual advantage. Firstly, it allows us to select a specific wavelength within the excitation spectrum, optimizing our outcomes. Secondly, our device can be adapted for use by diverse research teams working with different fluorescent proteins, offering the flexibility for customization based on their specific needs. Lastly, and no less importantly, even with advanced ambient light blocking measures in place, we remained committed to minimizing the detection of excitation light, thus guaranteeing that the light captured by our photosensitive component originates exclusively from the fluorescent proteins themselves.
To accomplish these goals, we used 3D printing, Arduino board, and ESP32 as our materials, and we preserved a chamber for the cuvette. These materials are small and not too hard to obtain. Considering the effect of receiving fluorescence, we came up with two kinds of arrangement. One device incorporates a 90-degree arrangement between the light source, sample chamber, and photoresistor, whereas the other has a 180-degree arrangement.
We conducted several tests to ensure the best measurements of our device. Firstly, we conducted FP light intensity tests to understand the best wavelengths for measurement, as well as optimizing our device in the process. Next, we added our engineered bacteria (pSB1C3-phla-mCerulean-p3-SaeRS) and Zn2+ into our variants of measurement, where we found correlation between Zn2+ concentration and fluorescent intensity.
After conducting tests with both the 90-degree and 180-degree configurations, we have learned that stability is a critical factor in determining the success of this FP device. As a result, the 90-degree configuration proves to be a better choice. Additionally, we have recognized the need for the development of a more efficient measurement method. Both the Arduino board and the associated driver code should be subject to further refinement to enhance measurement speed, precision and stability.The following URL is the link to our final device, which had made some improvement to enhance the performance of our device: Hardware page.
We took several requirements into consideration when designing our device. First and foremost, we would like to build a small, portable device that works as a fast test kit. In addition, due to the fact that the EET system operates only under anaerobic conditions, we need to be able to contain an anaerobic condition in our bacteria component. Furthermore, we hope that our measurements can be sensitive and accurate.
With these goals in mind, we adopted 3D printing, CoverWell Perfusion Chamber, syringe, screen-printed three-electrodes as our materials. These materials are smaller and cheaper than their conventional counterparts- an electrolyte reservoir and three electrode rods. CoverWell Perfusion Chamber provides an anaerobic environment, while screen-printed three-electrodes provides sensitive detection.
For the testing of our device, we connected our three-electrode system to the potentiostat. We first conducted a standard substance KCl test, to ensure our electrode system is working. Next, we loaded different solutions containing differing concentrations of Zn2+ ions. Our results are documented on CV (Cyclic Voltammetry) plots, for further interpretation.
After testing our device, we found several points we need to address and improve. First of all, we used Ag as our reference electrode, but after measuring, we found that our CV plots had noise peaks when running cycles on the potentiostat. After consulting with the teacher, he suggested we could change our Ag electrode into AgCl electrode in the future to optimize our results. Next, due to the fact that we need to run three to four cycles on potentiostat to have usable measurements, it can be timely for our users. To improve this situation, we could change our measurement methods and focus on electrical current values when applying -1V voltage, to optimize user experience.
If you wish to learn more about our EET device, please follow the link to our Hardware page.
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