iGEM 2023 — Team nycu-taipei

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# Notebook

### June

### July week 1& 2

### July week 3& 4

### August week 1& 2

### August week 3& 4

### September week 1& 2

### September week 3& 4

### October week 1& 2

Wet lab

Lab training for all members. Lab meeting entails biosafety training, lab
emergency prevention measures, machine operation protocols, and biological
waste management.

Dry lab

##### FP

We designed a device that can detect changes in fluorescent intensity.

HP

We discussed our approach and plan for human practice.

Wiki

In preparation for designing our wiki website, we learned CSS coding format.

Wet lab

##### EET

We reviewed papers on how to regulate the electron transfer system and
evaluated the possibility of constructing conductive E-pili in *E. coli*.

##### TCS

We retrieved the plasmid for the promoter *phla* and the two-component gene *saeRS* from Professor Bae and transformed them into *E. coli* DH5a.

Dry lab

##### FP

We designed a device that can transduce the signal of changes in fluorescent intensity detected by our device to the computer.

HP

We decided on the list of teams we wanted to collaborate with. Meanwhile, we
worked on fundraising for our team.

Wiki

In preparation for designing our wiki website, we learned the HTML5 coding
format.

Wet lab

##### Biomarker

We combed through the chemical biomarkers from the database markerDB, and narrowed it down by nonhereditary diseases. We then searched every biomarker for detection pathways.

##### EET

We looked for methods to construct Extracellular Electron Transfer pathway into *E. coli*.

##### TCS

We extracted plasmids which the genes two-component system *saeRS*, the fluorescent protein mCerulean, and the promoter *phla* are on.

Dry lab

##### FP

We built an Arduino code for the control panel of our fluorescent detection device.

##### EET

We designed a device that can detect electric signals from extracellular electron transfer signaling pathways.

HP

We visited the Asian Biotechnology Exhibition and interviewed biotechnology
companies about probiotic bacteria and diagnostic biomarkers.

Wiki

In preparation for designing our wiki website, we learned JavaScript and
ECMAScript coding format.

Wet lab

##### Biomarker

We narrowed down to 33 biomarkers from the database markerDB.

##### EET

We prepared competent cells and solutions for our experiments.

##### Hesperetin

*S.avermitilis* has the gene to produce rhamnosidase. We tried using liquid culture to extract the genomic DNA of *S.avermitilis* to clone the gene for Rhamnosidase.

##### TCS

We used PCR to amplify and retrieve the DNA sequence we needed from the plasmids' DNA. The genes we retrieved include gene *saeRS*, promoter *p3*, promoter *phla*, and gene *mCerulean*. We modified the program of the PCR until desired results were produced.

Dry lab

##### FP

We learned how to operate a 3D printing machine. And we created a 3D model of the device and configured the parameters based on the material we used, the machine specifications, and our device design.

HP

We decided to interview two expert physicians: one specialized in immuno-rheumatology, Dr. Chang Yen-An, and the other, Dr. Hsih Mo-Song, in neurology.

Wiki

In preparation for designing our wiki website, we learned the Document
Object Model (DOM), the Browser Object Model (BOM), AJAX (Asynchronous
JavaScript and XML), and how to use jQuery.

Wet lab

##### Biomarker

We narrowed down to 5 biomarkers from the database markerDB.

##### EET

We extracted the plasmids of *mtrCAB*.

##### FP

We measured the growth curve of the engineered *E. coli* carrying the
mCerulean fluorescent gene.

##### Hesperetin

We failed repeatedly because of the excessive amount of starch in the culture broth used in culturing *S.avermitilis*. So we switched over to solid media to culture *S.avermitilis*. However, we encountered another issue with extracting the genomic DNA of *S.avermitilis*, the DNA we managed to extract was broken into small fragments. To solve this problem, we changed the experiment protocol to using phenol and chloroform to purify the genomic DNA instead of using the Presto™ Mini gDNA Bacteria Kit.

##### TCS

We used PCR to amplify and retrieve the DNA sequence we needed from the plasmids' DNA. The genes we retrieved include *saeRS*, promoter *p3*, promoter *phla*, and gene *mCerulean*. We modified the program of the PCR until desired results were produced.

Dry lab

##### FP

We worked on optimizing our printing results and assembled our device. We also built a function-fitting Matlab code for our device modeling.

##### EET

We designed an electrical circuit for our EET detection device.

Wiki

We implemented a responsive layout and set up a two-column layout pattern
for our wiki website. In these two weeks, we also decided on our color
scheme and created templates for our wiki website's homepage, members, and
timeline pages.

Wet lab

##### Biomarker

We decided on L-Tyrosine as a new biomarker.

##### EET

To construct the EET reporting system into our *E. coli*, we extracted the genomic DNA of MG1655 for the gene *ccmA-H* and constructed a plasmid with it.

##### FP

We measured the growth curve of the engineered *E. coli* carrying the
mCerulean fluorescent gene.

##### TCS

We ligated the genes we needed for our construct design with Gibson Assembly, adjusting the proportion of each part until we succeeded in cloning our genes into an assembled plasmid.

Dry lab

##### FP

To optimize our fluorescent detection device, we visited TamKang University to discuss with Professor Hsu, Chun-Fei, who specializes in digital integrated circuit design. We modified our fluorescent detection device based on the professor’s advice.

##### EET:

We visited Professor Ta-Chung Liu from NYCU, who specializes in electrochemistry, to figure out the specifications of what circuit design and material would work best for our EET detection device.

HP

We attended the clubs and students’ association expo to share knowledge
about synthetic biology and our project. We also visited Dr. Chang Yen-An, a
physician specializing in immuno-rheumatology from Taipei Veterans General
Hospital, about the clinical demand for the type of diagnostic tool our team
aims to make.

Wiki

We produced the promotion video for our team project.

Wet lab

##### EET

We ligated the *mtrCAB* gene after the two-component system *saeRS* genes.

##### TCS

We transformed the engineered plasmid into our *E. coli*. Then we extracted the proteins that were produced by the *E. coli* and performed SDS-PAGE to check if the gene of interest is expressed. We also constructed two plasmid that contain the *phla* promoter and the *p3* promoter, both followed by the mCerulean gene, respectively.

Dry lab

##### FP

We built a new, modified version of our fluorescent detection device. We tested its function and ability and further modified the device accordingly. Also, we started collecting data for our device model.

##### EET

We designed a 3D model of the EET detection device.

##### TCS

We transformed the engineered plasmid into our E. coli. Then we extracted the proteins that were produced by the E. coli and performed SDS-PAGE to check if the gene of interest is expressed. We also constructed two plasmid that contain the phla promoter and the p3 promoter, both followed by mCerulean, respectively.

HP

We collaborated with the iGEM team from Chung Cheng University of Taiwan,
UGM of Indonesia, and UNC-Chapel Hill of the USA. We also visited Dr. Hsih Mo-Song, a
neurologist physician from Chang Gung Memorial Hospital, about the clinical
demand for the type of diagnostic tool our team aims to make. During this
period, we held a 17th-anniversary concert for all the NYCU-Taipei iGEM
alumni. We also held a synthetic biology seminar for NYCU undergraduates.

Wet lab

##### EET

We purified the proteins from our engineered *E. coli* and ran the SDS-PAGE to make sure the plasmid was constructed correctly. We also went to Tunghai University to visit Professor Li Shiue-Lin who specializes in electrochemistry and electromicrobiology to discuss the construction design and detection capability of our device.

##### TCS

We performed functional assay on our engineered *E. coli* to test the correlation between the concentration of zinc ions and the intensity of fluorescent protein produced by the *E. coli*.

Dry lab

##### FP

We tested our fluorescent detection device’s function and ability and modified the device accordingly. We also collected data to train our device model.

##### EET

We 3D printed our EET detection device and tested it with wet lab experiments.

HP

We organized, summarized, and reviewed the results of our year doing human practice.

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# Engineering

[Cycle 1: TCS + FP](#list-item-1)
[Design](#list-item-1-1)
[Build](#list-item-1-2)
[Test](#list-item-1-3)
[Learn](#list-item-1-4)
[Cycle 2: TCS + EET](#list-item-2)
[Design](#list-item-2-1)
[Build](#list-item-2-2)
[Test](#list-item-2-3)
[Learn](#list-item-2-4)
[Cycle 3: FP Device](#list-item-3)
[Design](#list-item-3-1)
[Build](#list-item-3-2)
[Test](#list-item-3-3)
[Learn](#list-item-3-4)
[Cycle 4: EET Device](#list-item-4)
[Design](#list-item-4-1)
[Build](#list-item-4-2)
[Test](#list-item-4-3)
[Learn](#list-item-4-4)

# Cycle 1: TCS + FP

![...](https://static.igem.wiki/teams/4822/wiki/wiki/engineering/engineering-tcs-fp.png)

# Design

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D) page.

We used three methods to ensure the success of the engineering:

1. We used the enzyme digestion method to check if the certain plasmid we constructed was accurate.
2. After successful transformation, we extracted and purified the protein produced by transformed *E. coli*, then used SDS-PAGE to check if the bacteria could produce a certain protein.
3. We measured fluorescent density to analyze the effectiveness of SaeRS.

# Build

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.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tcs-figure-2b-plasmid-map-of-psb1c3-phla-mcerulean-p3-saers-tag-plasmid.png )

Figure 1. The plasmid map of the pSB1C3-phla-mCerulean-p3-SaeRS plasmid.

# Test

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.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tcs-figure-1-enzyme-digestion-confirmation-of-gibson-assembly-results.png)

Figure 2. The result of enzyme digestion confirmation of the Gibson assembly results from different bacteria colonies. The numbers labeled above the wells are the assigned number of the bacteria colony. The bacteria of colony 16 carry the complete plasmid of pSB1C3-phla-mCerulean-p3-SaeRS 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.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tcs-figure-3-result-of-saes-sds-page.png )

Figure 3. The result of SDS-PAGE with the SaeS protein.

Last but not least, after the measurement of SaeRS function, we notice the noticeable correlation between fluorescent density and Zn2+ concentration.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/sae-zn-result-0-1.png)

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/sae-zn-result-1-1-4.png)

Figure 4. The result of SaeRS device(BBa\_K4822005+BBa\_K4822007) Zn2+ sensity test.

To obtain further details about the experiments and their outcomes, please refer to the [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page and the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

# Learn

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.

# Cycle 2: TCS + EET

![...](https://static.igem.wiki/teams/4822/wiki/wiki/engineering/tcs-eet.png)

# 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:

1. We used the enzyme digestion method to check if the certain plasmid we extracted was accurate.
2. After successful transformation, we extracted and purified the protein produced by transformed *E. coli*, then used SDS-PAGE to check if the bacteria could produce a certain protein.
3. We used a device to check the functionality and feasibility of the engineered *E. coli*.

# Build

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.

# Test

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.

# Learn

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

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.

# Cycle 3: FP Device

![...](https://static.igem.wiki/teams/4822/wiki/wiki/engineering/fp-cycle.png)

# Design

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.

# Build

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.

# Test

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.

# Learn

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D).

# Cycle 4: EET device

![...](https://static.igem.wiki/teams/4822/wiki/wiki/engineering/eet-cycle.png)

# Design

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.

# Build

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.

# Test

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.

# Learn

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D) page.

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# Experiments

[SaeRS
& Calprotectin](#list-item-1)
[Overview](#list-item-1-1)
[Preparation](#list-item-1-2)
[Plasmid Engineering](#list-item-1-3)
[Protein Function](#list-item-1-4)
[TyrR
& L-Tyrosine](#list-item-2)
[Overview](#list-item-2-1)
[Preparation](#list-item-2-2)
[Plasmid Engineering](#list-item-2-3)
[Protein Function](#list-item-2-4)
[Fluorescent
Protein](#list-item-3)
[Overview](#list-item-3-1)
[Plasmid Engineering](#list-item-3-2)
[Protein Function](#list-item-3-3)
[Device Testing](#list-item-3-4)
[α-Rhamnosidase](#list-item-4)
[overview](#list-item-4-1)
[Plasmid Engineering](#list-item-4-2)
[Protein Function](#list-item-4-3)
[Functional Assay](#list-item-4-4)

# SaeRS & Calprotectin

# Overview

The biomarker Calprotectin consists of protein subunits S100A8 and S100A9 that can bind to zinc ions (Zn2+). It is related to diseases like rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease.In order to construct an *E. coli* that can detect the biomarker Calprotectin, we adopted the Two-Component System(TCS) SaeRS from *Staphylococcus aureus*.

The SaeRS system of *Staphylococcus aureus* is composed of two proteins, SaeR and SaeS, which work together to detect environmental signals and regulate downstream gene expression. When the sensor histidine kinase SaeS phosphorylates the response regulator SaeR, the phosphorylated SaeR binds to a direct repeat sequence in specific downstream promoters, activating gene expressions. However, in the presence of Zn2+, the phosphorylation of SaeS is inhibited, preventing the phosphorylation of SaeR and consequently inhibiting downstream gene expression. There are studies that show when Calprotectin binds Zn2+, it could effectively prevent downstream gene expressions from being inhibited.[1] This implies that when Calprotectin is present, it would increase downstream gene expression.

We apply this mechanism to our experiment design. *Phla* is the promoter of the *hla* gene in *Staphylococcus aureus*. It is one of the promoters that is regulated by the two-component system SaeRS. The experiment concept we adopted is that under the presence of a higher concentration of Calprotectin, there would be fewer Zn2+ free ions. With fewer Zn2+ free ions, the expression of the gene for the fluorescent protein mCerulean would increase, producing a higher fluorescent intensity for us to detect. This way, we can infer the concentration of Calprotectin by observing the fluorescent density emitted by the *E. coli*.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/tcs-figure-1-experiment-design-zn2.png )

Figure 1. Experiment design.

# Preparation

We retrieved the plasmids for the promoter *phla* (pYJ-Phla-lacZ plasmid) and the two-component gene *saeRS* (pCL-sae-usa plasmid) from Professor Taeok Bae from the Department of Microbiology and Immunology, Indiana University School of Medicine-Northwest, USA. We transformed the two plasmids into *E. coli* DH5ɑ, respectively, to amplify them. The bacteria were cultured in LB broth with antibiotic Ampicillin. We extracted the aforementioned two plasmids which contain the *phla* promoter and the gene *saeRS*, and we also extracted plasmids that contained the gene for the fluorescent protein mCerulean.

We used Polymerase Chain Reaction (PCR) to amplify specific DNA sequences we needed for the new plasmid DNA construction. These gene fragments include gene *saeRS*, promoter *p3*, promoter *phla*, and the gene for mCerulean. In the beginning, we used the typical PCR programs for each fragment, but the amplification results were unsuccessful after several tries. Therefore, we conducted gradient PCR to discover the favorable temperature condition. We employed the adjusted program settings (see more at [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)) based on the gradient PCR results and ultimately obtained the desired PCR products. Later, electrophoresis was done to verify each DNA fragment length and we conducted gel extraction to retrieve them for further experiments.

# Plasmid Engineering

We ligated the five genes, *saeRS*, Terminator, Origin+CmR, *phla*, and the gene for mCerulean, for our construct design using Gibson Assembly and transformed the engineered plasmid into *E. coli*. We adjusted the proportion of each part until we succeeded in cloning our genes into an assembled plasmid.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/tcs-figure-2-plasmid-map-of-psb1c3-phla-mcerulean-p3-saers-tag-plasmid.png )

Figure 2. Plasmid map of the pSB1C3-phla-mCerulean-p3-SaeRS plasmid.

To ensure that we have successfully assembled all parts of our genes and that the *E. coli* can express the genes of interest, we extracted the proteins that were produced by the *E. coli* and performed Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis(SDS-PAGE).

After the first SDS-PAGE result, we observed that the concentration of SaeS is extremely low. Therefore, we designed another plasmid, on which the promoter of saeRS is BBa\_J23100, a constitutive promoter with high transcription levels. Meanwhile, we also plan to point mutate on the XbaI digestion site, and add a His tag on SaeR.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/tcs-figure-3-plasmid-map-of-psb1c3-phla-mcerulean-bba-j23100-mut-saers-2tag.png)

Figure 3-1. Plasmid map of the pSB1C3-phla-mCerulean-BBa\_J23100-mutSaeRS plasmid.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/img-3112.jpeg)

Figure 3-2. *saeR* point mutation.

# Protein Function

To determine the sensitivity of our engineered *E. coli*, we designed a functional assay to test it out. We cultured the engineered *E. coli* in various concentrations of Zn2+ and measured the fluorescent intensity and OD600 of the *E. coli*. The observed fluorescent density of the *E. coli* allowed us to assess the relationship between Zn2+ concentration and the expression level of the fluorescent genes..

# TyrR & L-Tyrosine

# Overview

L-Tyrosine is an essential amino acid in our bodies and it is associated with inflammation in patients with Lewy Body Dementia (LBD). In order to detect the presence of L-Tyrosine with our engineered *E. coli*, we introduced the usage of *TyrR* protein, a DNA-binding transcriptional dual regulator. The gene tyrR exists in *E. coli* K-12 MG1655, allowing it to produce the TyrR protein. TyrR has several binding sites for multiple substrates and could act as an activator or a repressor under different circumstances. In the presence of L-Tyrosine, TyrR can self-associate into hexamers that bind to both the strong box and weak box of the promoter, thereby influencing downstream gene expressions.

The promoter we selected was *paroF*, which is repressed in the presence of L-Tyrosine. Two TyrR protein hexamers are involved in this reaction. One of the TyrR hexamers can identify the TyrR box 1(strong box) and TyrR box 2(weak box), and then binding across them. Another hexamer binded to the upstream TyrR box 3(strong box) and the adjacent region.

Following the promoter *paroF* is the gene encoding the fluorescent protein mRFP1. The fluorescence emission intensity could indicate the degree of *paroF* repression and, subsequentially, the concentration of L-Tyrosine.

# Preparation

We received the *E. coli* carrying the pLacUV5-tyrR-paroF-mRFP1 plasmid from Professor Yi-Chun Yeh from the Department of Chemistry, National Taiwan Normal University, Taiwan. However, this plasmid bears the promoter *pLacUV5*, which is an unnecessary inducible promoter for our experiment design. Therefore, we decided to replace it with a constitutive promoter.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/l-tyrosine-ntnu-agar-plate.jpg)

Figure 4. *E. coli* DH5ɑ with pLacUV5-tyrR-paroF-mRFP1 plasmid. The agar plate was provided by Prof. Yi-Chun Yeh.

The pET-21a(+) plasmid was selected as the new backbone of tyrR-paroF-mRFP1 due to the presence of the constitutive T7 promoter in its sequence. It was then transformed into *E.coli* DH5ɑ and cultured on LB + Ampicillin agar plates. Colonies were picked and cultured in LB broth containing the antibiotic Ampicillin to confirm the success of the transformation.

# Plasmid Engineering

Both the pLacUV5-tyrR-paroF-mRFP1 plasmid and the pET-21a(+) plasmid were digested with EcoRI and XhoI enzymes, followed by electrophoresis and gel extraction to obtain the desired DNA fragments. For PLacUV5-tyrR-ParoF-mRFP1 plasmid, the desired length would be 2496 base pairs, while for pET-21a(+) plasmid, the correct length would be 5409 base pairs.

We ligated both DNA fragments with T4 DNA ligase and left at room temperature for one hour. We tried two molar ratios of vector DNA to insert DNA, specifically 1:3 and 1:1, to determine a better recipe for DNA ligation. The ligated product was then transformed into *E. coli* DH5ɑ and cultured on LB + Ampicillin agar plate.

# Protein Function

The engineered *E.coli* bacteria were transferred from LB + Ampicillin broth to M9 minimal broth, which was supplemented with a 0.4% glucose solution, 2 mM MgSO4 solution, 0.1 mM CaCl2 solution, 0.5 μg/mL Thiamine hydrochloride, and 100 μg/mL Ampicillin. The L-Tyrosine concentration was varied in a gradient from 0 μM to 100 μM, with intervals of 10 μM.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/l-tyrosine-gradient-concentration-tube-photo.jpg)

Figure 5. The gradient of L-Tyrosine concentration in supplemented M9 minimal broth. Engineered *E. coli* was cultured in the 37℃ incubator.

We measured the fluorescent intensity and OD600 from the engineered E. coli to verify the function of our detection device. The fluorescent density of Tyrosine biosensor decreased as the L-Tyrosine concentration increased. We concluded that our biosensor has the ability to detect the existence of L-Tyrosine. In the end, we examined whether our constructed biosensor could provide a rough estimate of the L-Tyrosine concentration in the supplemented M9 minimal broth by analyzing the fluorescent density data.

# Overview

MtrA, MtrB, and MtrC are the major c-type cytochromes from *Shewanella oneidensis* involved in the extracellular electron transfer (EET) pathway, serving as electron carriers and helping the electron transfer. The CcmA-H proteins are necessary for the maturation of MtrA, MtrB, and MtrC cytochromes. In order to construct an EET pathway as one of the reporting systems in our project, we need to co-transform the pSB1C3-phla-(MtrCAB+tag)-Sae plasmid and the pYJ\_BBa\_J23100\_CcmAH\_BBa\_B0015 plasmid which carries the gene *ccmA-H* into *E. coli*. [5][6]

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/eet-figure-1-eet-pathway.png)

Figure 6. Extracellular Electron Transfer Pathway.

# Plasmid Engineering

# pSB1C3-phla-(MtrCAB+tag)-Sae plasmid construction

We purchased the pETSXM2-pLacI-mtrCAB plasmid from Addgene. To connect with the upstream biomarker detection system, we replaced the mCerulean gene on the *saeRS* plasmid with the *mtrCAB* on the plasmid pETSXM2-pLacI-mtrCAB. We used PCR to amplify the *mtrCAB* part and added one of two restriction enzyme cutting sites, SacII and BamHI, to each end. Then, we used enzyme digestion to obtain the *mtrCAB* fragment and *saeRS* plasmid excluding the mCerulean gene. We ligated these two fragments together and completed pSB1C3-phla-(MtrCAB+tag)-Sae plasmid construction. (see more at [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D))

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/eet-figure-2-gene-map-of-psb1c3-phla-mtrcab-tag-sae-plasmid.png)

Figure 7. Plasmid map of the pSB1C3-phla-(MtrCAB+tag)-Sae plasmid.

# pYJ\_BBa\_J23100\_CcmAH\_BBa\_B0015 plasmid construction

The *ccmA-H* gene on the pYJ\_BBa\_J23100\_CcmAH\_BBa\_B0015 plasmid is necessary for cytochrome maturation. Cytochrome maturation is a post-translational process involving covalent attachment of heme to the apocytochrome polypeptide by two thioether bonds. We extracted the gDNA of *E. coli* K-12 MG1655 and used PCR technique to amplify the *ccmA-H* part of the gDNA. Then we conducted enzyme digestion and ligation to combine *ccmA-H* genes with the replication origins, the Ampicillin resistant gene, the promoter BBa\_J23100, the ribosome binding site BBa\_B0034, and the terminator from the pSB1C3-mCerulean plasmid.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/eet-figure-3-gene-map-of-pyj-bba-j23100-ccmah-bba-b0015-plasmid.png)

Figure 8. Plasmid map of the pYJ\_BBa\_J23100\_CcmAH\_BBa\_B0015 plasmid.

# Transformation

After we completed the construction of both plasmids above, we had to co-transform them
into
*E. coli* strain BL21 with the aim of obtaining expression strain.

# Protein Function

# Protein Purification

To check if our constructed genes are expressed as expected, we induced *E. coli* to produce the protein, and then tested their performance properties. In the previous primer design, we added 6x his tag after our target genes *mtrCAB* and *ccmA-H*(we only added his tag after the *mtrB* and *ccmH* gene due to other genes being connected to each other). Thus, there will be 6 histidine at the end of the two proteins. Since histidine has a high affinity to the metal ion, Ni2+, we used Ni-column to catch the proteins with histidine with the aim to purify the protein product. (see more at [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D))

# SDS-Page

After we purified the protein product, we needed to check whether the protein's properties were as we expected. We know from reviewing databases that the molecular weight of the MtrB protein is about 77kDa, and the CcmH protein is about 39kDa. By running SDS-Page, we can measure the mass of the protein and know if the *E. coli* can successfully synthesize the desired protein.(see more at [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D))

# Protein Assay

# Heme staining

To check if our cytochrome, MtrA, MtrB and MtrC, are being synthesized in the engineered *E.coli*, we conducted heme staining, which is an additional staining of SDS-Page. The gel is immersed in the 3,3',5,5'-Tetramethylbenzidine (TMBZ) solution, where the TMBZ will bind to the heme in the cytochromes. After adding hydrogen peroxide into the solution, the TMBZ-bound heme will turn bluish-green (λmax: 655 nm), therefore allowing us to see the cytochromes.
(see more
at [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D))

# Device Testing

We built up a simple device to check the functionality and feasibility of our engineered *E. coli*. We injected the engineered *E. coli* into a CoverWell Perfusion Chamber. We utilized the three-electrode system which consisted of two carbon electrodes, a working electrode and a counter electrode, one silver electrode as a reference electrode, and cyclic voltammetry to test the current intensity. For more details, see the [dry lab device](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D) page. [7]

# α-Rhamnosidase

# Overview

Hesperetin is a flavonoid compound, commonly found in citrus fruits, particularly in the peels of oranges, grapefruits, and lemons. It has many functions that benefit our health, like antioxidants, anti-inflammatory, anticancer, lipid-lowering, antidiabetic, etc. It can be produced by cleaving rhamnose and glucose glycosides from hesperidin by α-Rhamnosidase and β-Glucosidase.

Typically, humans absorb the precursor hesperidin and convert it into hesperetin using α-Rhamnosidase and β-Glucosidase enzymes within our bodies. However, the challenge for our bodies is that the amount of α-Rhamnosidase in our bodies varies among individuals. Even if someone consumes a lot of hesperidin, they may not be able to synthesize a comparable amount of hesperetin. And because α-Rhamnosidase is the rate-determining step in the reaction, which transforms hesperidin transfers to hesperetin, we chose to design the gene for α-Rhamnosidase into our engineered plasmid. The ultimate goal is to raise the quantity of α-Rhamnosidase in our bodies and help relieve inflammation responses.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/pathways-for-enzymatic-deglycosylation-of-hesperidin-to-yield-hesperetin-via.png)

Figure 9. Pathways for enzymatic deglycosylation of hesperidin to yield hesperetin. [8]

# Plasmid Engineering

# Preparation of genomic DNA

α-Rhamnosidase is an enzyme responsible for cleaving rhamnose from hesperidin. The bacteria *Streptomyces avermitilis* carries the Rhamnosidase gene in its genomic DNA. Therefore, we employed this conversion method for this specific part of our project.

# Polymerase chain reaction(PCR)

We conducted PCR to amplify the Rhamnosidase gene sequence. Additionally, by adding a His tag to the sequence, it is more convenient for us to identify the rhamnosidase protein in the subsequent experiments.

# Plasmid construction + Transformation

We used the pSB1C3 plasmid as the backbone in our plasmid construct. We employed the restriction enzyme sites HindIII and BamHI to insert the gene for the Rhamnosidase into our plasmid. We transferred the engineered plasmid into *E. coli* DH5α to amplify the plasmid. We cultured the E. coli in LB + Cm to confirm the success of the transformation.

# Protein Function

# Protein expression

We transformed the plasmid into *E. coli* BL21 to express Rhamnosidase.

# Protein purification + SDS page

We Used Ni-NTA to purify protein, then ran the SDS-PAGE to check if the *E. coli* expresses the Rhamnosidase protein.

# Functional Assay

# Test Rhamnosidase function (*in vitro*)

We conducted in vitro experiments, applying the extracted Rhamnosidase to facilitate the conversion of hesperidin to hesperetin.

# Test Rhamnosidase function (in vivo)

The objective of this experiment is to evaluate the conversion efficiency of hesperidin after feeding mice with *E.coli* containing the Rhamnosidase gene and hesperidin simultaneously. We plan to analyze the levels of hesperetin and hesperidin in their blood.

# References

1. [Liu, Q., Yeo, W. S., & Bae, T. (2016). The SaeRS Two-Component System of Liu, Q., Yeo, W. S., & Bae, T. (2016). The SaeRS Two-Component System of Staphylococcus aureus. *Genes, 7*(10), 81.](https://doi.org/10.3390/genes7100081)
2. [Pittard, J., Camakaris, H., & Yang, J. (2005). The TyrR regulon. *Molecular microbiology*, 55(1), 16–26.](https://doi.org/10.1111/j.1365-2958.2004.04385.x)
3. [Cobbett C. S. (1988). Repression of the aroF promoter by the TyrR repressor in Escherichia coli K-12: role of the 'upstream' operator site. *Molecular microbiology, 2*(3), 377–383](https://doi.org/10.1111/j.1365-2958.1988.tb00042.x)
4. [Lin, C., Jair, Y. C., Chou, Y. C., Chen, P. S., & Yeh, Y. C. (2018). Transcription factor-based biosensor for detection of phenylalanine and tyrosine in urine for diagnosis of phenylketonuria. Analytica chimica acta, 1041, 108–113.](https://doi.org/10.1016/j.aca.2018.08.053)
5. [Jensen, H. M., Albers, A. E., Malley, K. R., Londer, Y. Y., Cohen, B. E., Helms, B. A., Weigele, P., Groves, J. T., & Ajo-Franklin, C. M. (2010). Engineering of a synthetic electron conduit in living cells. *Proceedings of the National Academy of Sciences of the United States of America, 107*(45), 19213–19218.](https://doi.org/10.1073/pnas.1009645107)
6. [Su, L., Fukushima, T., Prior, A., Baruch, M., Zajdel, T. J., & Ajo-Franklin, C. M. (2020). Modifying Cytochrome c Maturation Can Increase the Bioelectronic Performance of *Engineered Escherichia coli. ACS synthetic biology, 9*(1), 115–124.](https://doi.org/10.1021/acssynbio.9b00379)
7. [Su, L., Fukushima, T., & Ajo-Franklin, C. M. (2020). A hybrid cyt c maturation system enhances the bioelectrical performance of engineered Escherichia coli by improving the rate-limiting step. *Biosensors & bioelectronics, 165*, 112312.](https://doi.org/10.1016/j.bios.2020.112312)
8. [Piñuel, L., Breccia, J. D., Guisán, J. M., & López-Gallego, F. (2013). Production of hesperetin using a covalently multipoint immobilized diglycosidase from Acremonium sp. DSM24697. J*ournal of molecular microbiology and biotechnology, 23*(6), 410–417.](https://doi.org/10.1159/000353208)

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# Results

[Two component system](#list-item-1)
[Plasmid Engineering](#list-item-1-1)
[Protein function](#list-item-1-2)
[L-Tyrosine](#list-item-2)
[Plasmid Engineering](#list-item-2-1)
[Protein Function](#list-item-2-2)
[Extracellular Electron Transfer Pathway](#list-item-3)
[pSB1C3-phla-(MtrCAB+tag)-Sae plasmid construction](#list-item-3-1)
[pYJ\_BBa\_J23100\_CcmAH\_BBa\_B0015 plasmid](#list-item-3-2)
[Project Achievements](#list-item-4)
[What we did achieve](#list-item-4-1)
[What we didn’t achieve](#list-item-4-2)

# Two component system

# Plasmid Engineering

After we transformed the Gibson Assembly ligation product into *E. coli*, we cultured the transformed bacteria on solid media with antibiotic Chloramphenicol(Cm). In order to confirm our ligation result, we picked several colonies and amplified them in liquid LB + Cm media to perform plasmid extraction. From Figure 1, we see that colony 16 had the desired complete plasmid.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tcs-figure-1-enzyme-digestion-confirmation-of-gibson-assembly-results.png )

Figure 1. The result of enzyme digestion confirmation of the Gibson assembly results from different bacteria colonies. The numbers labeled above the wells are the assigned number of the bacteria colony. The bacteria of colony 16 carry the complete plasmid of pSB1C3-phla-mCerulean-p3-SaeRS plasmid.

We cultured bacteria from colony 16 in liquid media to double-confirm our result. From Figure 2b, we can be certain that we successfully constructed the pSB1C3-phla-mCerulean-p3-SaeRS plasmid and transformed it into *E. coli*.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tcs-figure-2a-enzyme-digestion-confirmation-of-the-colony-16-plasmid-construction.png )

Figure 2a. The result of enzyme digestion confirmation of the colony 16 plasmid construction. The wells labeled “S” are the pSB1C3-phla-mCerulean-p3-SaeRS plasmid (Fig. 2b), while wells labeled “C” are the pSB1C3-J364007\_Promoter\_RBS\_T1\_T2\_with\_mCerulean\_(hGFP)\_CDS plasmid (Fig. 2c). The C groups serve as a comparative reference to ensure the functionality of the digestive enzymes.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tcs-figure-2b-plasmid-map-of-psb1c3-phla-mcerulean-p3-saers-tag-plasmid.png )

Figure 2b. The plasmid map of the pSB1C3-phla-mCerulean-p3-SaeRS plasmid.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tcs-figure-2c-psb1c3-j364007-promoter-rbs-t1-t2-with-mcerulean-hgfp-cds-plasmid.png )

Figure 2c. The plasmid map of the pSB1C3-J364007\_Promoter\_RBS\_T1\_T2\_with\_mCerulean\_(hGFP)\_CDS plasmid.

To assemble the pSB1C3-phla-mCerulean-BBa\_J23100-SaeRS plasmid, we are in the process of cloning three DNA fragments through PCR and subsequently ligating them using Gibson assembly. Currently, we have completed the PCR for one fragment, and the construction of the pSB1C3-phla-mCerulean-p3-Sae plasmid (BBa\_K4822005+BBa\_K4822007). Our experiment is still ongoing.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/sae-figure-4-j23100-sae-pcr.png)

Figure 3. The Sae\_mut\_f + Saeplasmid\_r primer pair PCR product for J23100-Sae plasmid construction.

We extracted protein from the successfully transformed bacteria and performed SDS-PAGE. Due to our primer design, we only added one His tag after the SaeS protein in our plasmid construct. Since the genes for SaeR and SaeS are situated downstream of the same promoter and the two genes adjoin, confirming the expression of one protein is to ensure the expression of the gene *saeRS*. Therefore, we only performed protein purification and SDS-PAGE on the SaeS protein. From Figure 4, we can see that the bacteria expressed the *saeRS* gene and produced the protein.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tcs-figure-3-result-of-saes-sds-page.png )

Figure 4. The result of SDS-PAGE with the SaeS protein.

# Protein function

To investigate the sensitivity of our Sae plasmid device, we conducted fluorescent intensity and OD600 measurements to investigate the relationship between fluorescent density and Zn2+ concentration. We performed the Zn2+ test twice, eliminating data with OD600 measurements below 0.2 to mitigate significant errors caused by the limited bacterial numbers. We assume that the growth problem in high Zn2+ concentration groups is due to the high osmotic pressure. We noted that for Zn2+ concentrations below 1mM, the trend in fluorescent density is not distinct. However, within the range of 1 to 1.4mM Zn2+ concentration, there is a notable correlation between fluorescent density and Zn2+ concentration. However, our experiment only measured fluorescent density in Zn2+ concentrations up to 1.4 mM. We plan to conduct more experiments to collect more data to determine the sensitivity of our plasmid device.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/sae-zn-result-0-1.png)

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/sae-zn-result-1-1-4.png)

Figure 5. The result of the SaeRS device(BBa\_K4822005+BBa\_K4822007) Zn2+ sensitivity test.

# L-Tyrosine

# Plasmid Engineering

After obtaining the desired DNA fragments, two molar ratios of vector DNA and insert DNA were implemented, 1:1 and 1:3, respectively, to determine a better recipe for successfully ligating these two fragments.

Upon examining the results of transformation and culture, we observed that LB + Ampicillin agar plates with a 1:3 ratio had multiple colonies grown, whereas plates with a 1:1 ratio did not yield any colonies.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/l-tyrosine-ligation-ratio-comparison.jpeg)

Figure 6. Agar plate result of ligation using two different ratios. Plate on the left side was 1:3 ratio. Plate on the right side was 1:1 ratio.

After receiving the results of ligation, we proceeded to confirm the length of the constructed plasmid. We chose two restriction enzyme sites, BamHI and NdeI, which would generate distinct DNA fragment lengths that we could differentiate. Two colonies were selected randomly, each of which went through the aforementioned process.

The results were analyzed using agarose gel electrophoresis. Colony 1 and Colony 2 were divided into two groups and subjected to restriction enzyme digestion using BamHI and NdeI, respectively. After cutting with two BamHI sites, the DNA fragment lengths should be 5412 and 2493 base pairs. When cut with two NdeI sites, the DNA fragment lengths were 6052 and 1853 base pairs. The confirmation results indicated that both Colony 1 and Colony 2 contained the desired plasmid. Consequently, we proceeded with Colony 1 for the subsequent experiment.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/tyrr-figure-colony-1-2-digestion-confirmation.png )

Figure 7. The electrophoresis results from restriction enzyme digestion products of two different colonies. C1 represents Colony 1 and C2 represents Colony 2.

# Protein function

We measured the correlation between fluorescent density and Tyrosine concentration. Correlation coefficient R2 is 0.8542, which signifies notable correlation. This result shows that our TyrR device is applicable, but there is still room for improvement

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/l-tyr-plotting-graph.png)

Figure 8. The plotting graph of the correlation between fluorescent density and Tyrosine concentration.

# Extracellular Electron Transfer Pathway

# pSB1C3-phla-(MtrCAB+tag)-Sae plasmid construction

After we received pETSXM2-pLacI-mtrCAB plasmid from Addgene, we conducted enzyme digestion to check the correctness of this plasmid. We use restriction enzymes XhoI and BamHI to digest the plasmid DNA. Only one cutting site enzyme digestion allowed us to check the total length of pETSXM2-pLacI-mtrCAB plasmid while several enzyme cutting site digestion allowed us to check if it is the correct plasmid DNA. By analyzing the gene map (Figure 9.), we predicted that the result of digestion by XhoI and BamHI would have two bands at 6.7k and 2.3k. Also, there is a chance that our pETSXM2-pLacI-mtrCAB plasmid hasn't been digested completely. So, the band at 9k still showed up. From the electrophoresis result (Figure 10.), we can learn that this plasmid DNA is correct.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/eet-figure-1-purchased-mtr-plasmid.png)

Figure 9. Plasmid map of the pETSXM2-pLacI-mtrCAB plasmid.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/eet-figure-2-red-of-petsxm2-placi-mtrcab-plasmid.png)

Figure 10. The result of enzyme digestion confirmation of the pETSXM2-pLacI-mtrCAB plasmid.

Afterward, we kept extracting pETSXM2-pLacI-mtrCAB plasmid to obtain enough amount of plasmid to do the Polymerase Chain Reaction(PCR) to amplify *mtrCAB* fragments and add on two cutting sites, SacII and BamHI.

Then, we use SacII and BamHI to digest both vector DNA, pSB1C3-phla-Sae plasmid, and *mtrCAB* fragment. By running electrophoresis (Figure 11.), we can use gel extraction to obtain purified DNA fragments. After that, we did ligation to make them a complete plasmid. Lastly, we transformed the modified plasmid into *E. coli* DH5α. We cultured the bacteria on the LB agar plate with chloramphenicol as a selection marker.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/eet-figure-3-restriction-enzyme-digestion-result-of-mtrcab-pcr-product-and-vector-dna.png)

Figure 11. The result of enzyme digestion confirmation of the mtrCAB PCR product and the vector DNA.

After the bacteria survived on the LB agar plate with chloramphenicol. We have to check whether the bacteria is harboring the correct plasmid. We extracted plasmid DNA from it and digested the plasmid DNA with a restriction enzyme, XbaI. From the gene map, it is clear that we can obtain 3 fragments after digesting by the restriction enzyme XbaI. The size of each fragment will be about 5k, 3k, 1.5k. The electrophoresis results of colony 1 and colony 3 can fit in the prediction and prove our plasmid construction successfully. (Figure 12.)

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/eet-figure-4-plasmid-dna-extracted-from-transformation-strain.png)

Figure 12. The result of enzyme digestion confirmation of the plasmid DNA extracted from transformation strain. (U: Uncut, C: Cut by XbaI)

# pYJ\_BBa\_J23100\_CcmAH\_BBa\_B0015 plasmid

The *ccmA-H* gene cluster is taken from the *E. coli* genome. We choose BBa\_J23100 and BBa\_B0034 as promoter and RBS (p-r), and BBa\_B0015 as terminator. Moreover, we picked replication origin and Ampicillin resistance gene (O-A) from pYJ-phla-LacZ to make the engineered bacteria selectable with LB+Chloramphenicol+Ampicillin medium. In these fragments, *ccmA-H,* p-r, and O-A are prepared by PCR, and BBa\_B0015 is prepared from pSB1C3-mCerulean by enzyme digestion. One problem we faced is that p-r fragments are too short to stain clearly under UV light. Therefore, we prepared more than 30 times p-r PCR product volume, then concentrated 30X to see the band while cutting the gel. Another problem we faced is the ligation of this plasmid. Because running out of time, we are not able to finish this experiment before the wiki freezes. We tried several rates of concentration, but could not get the right plasmid before we ran out of PCR products and polymerase.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/result/eet-figure-5-electrophoresis-of-ccm-pcr.png)

Figure 13. The result of electrophoresis of our PCR product.

![...](https://static.igem.wiki/teams/4822/wiki/wiki/experiment/eet-figure-3-gene-map-of-pyj-bba-j23100-ccmah-bba-b0015-plasmid.png)

Figure 6. Plasmid map of pYJ-BBa\_J23100-CcmAH-BBa\_B0015

# Project Achievements

# What we did achieve

1. Construction of sae device to detect Zn2+, and report by mCerulean fluorescent density.
2. Construction of the Tyr device to detect Tyrosine, and report by mRFP1 fluorescent density.
3. Design two plasmids for EET pathway construction.

# What we didn’t achieve

1. Construction of BBa\_J23100-*saeRS* device.
2. Extraction of *Streptomyces avermitilis* gnomic DNA.
3. Construction of pYJ\_BBa\_J23100\_CcmAH\_BBa\_B0015 plasmid.
4. Co-transformation of pSB1C3-phla-(MtrCAB+tag)-Sae plasmid and pYJ\_BBa\_J23100\_CcmAH\_BBa\_B0015 plasmid into the *E. coli* BL21.

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