iGEM 2022 — Team dku-china

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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Engineering

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Our project went through two separate lines of engineering, with each line containing engineering DBTL cycles. The following is a record of the two lines we followed.

## Line 1: Engineering the csoR-Based System

#### *Cycle 1 (Stage 1) — Design*

We found information regarding the csoR-Based System from a paper [1], which gave the genetic sequence of A. caldus csoR and introduced its role in A. caldus’s copper detoxification system. The paper identified three possible promoters, respectively named PI, PII, and PIII, for csoR. Although the paper concluded that PII is the copper-sensitive promoter, we suspected that PIII could also become copper-sensitive through adequate improvement. Thus we constructed the PIII-csoR-based system and began characterizing it as the first engineering cycle.

#### *Cycle 1 (Stage 2) — Build*

We characterized the csoR-Based System by creating transcription units with csoR and PIII. For more information regarding this, please refer to the protocols on our [Experiments](https://2022.igem.wiki/dku-china/experiments) page.

#### *Cycle 1 (Stage 3) — Test*

After testing csoR with 0 µM, 5 µM, 50µM, 0.5 mM, and 5 mM concentrations of copper, we verified that the PIII-csoR system is indeed not copper-sensitive. For more information regarding this, please refer to our [Results](https://2022.igem.wiki/dku-china/results) page.

#### *Cycle 1 (Stage 4) — Learn*

From the results, we learned that the original version of the PIII-csoR system does not work as expected, and therefore that further work must be done to alter the structure of PIII and allow the system to work properly.

#### *Cycle 2 (Stage 1) — Design*

We designed the improvement process of the PIII-csoR system. Since the main problem is with the copper sensitivity of PIII, we decided to improve the PIII-RBS combination via high-throughput experimentation.

#### *Cycle 2 (Stage 2) — Build*

Through random mutation and high-throughput experimentation, we eventually constructed 96 variants of PIII-RBS combinations (registered BBa\_K4494003-K4494098 with 78 unique sequences in total). All were characterized. To learn more about how the experimentation process was conducted, please refer to the protocols on our [Experiments](https://2022.igem.wiki/dku-china/experiments) page.

#### *Cycle 2 (Stage 3) — Test*

We conducted a thorough analysis of the 96 variants we got. For more information regarding this, please refer to our [Results](https://2022.igem.wiki/dku-china/results) page.

#### *Cycle 2 (Stage 4) — Learn*

Finally, through the 96 variants, we found several which are proven as functioning.

## Line 2: Engineering the CusR-Based System

#### *Cycle 1 (Stage 1) — Design*

We planned to construct a benchmark for our project and try to improve an existing part by characterizing and improving a part in the iGEM registry. After identifying several possible candidates, we eventually selected the CusR-Based System because of its proven superiority and rich documentation.

#### *Cycle 1 (Stage 2) — Build*

We mutated part BBa\_K1980004 and characterized both the original part and the mutants. To learn more about how the experimentation process was conducted, please refer to the protocols on our [Experiments](https://2022.igem.wiki/dku-china/experiments) page.

#### *Cycle 1 (Stage 3) — Test*

The part BBa\_K1980004 and its variants were tested with copper concentrations of 0 and 50µm. Eventually, the characterization results showed that none of the distinct variants were copper-sensitive, indicating that our initial goal has failed. However, we successfully produced a very strong promoter (registered BBa\_K4494102).

#### *Cycle 1 (Stage 4) — Learn*

From this experience, we understood how sometimes an unexpected discovery could occur in the process of scientific research. Although we were unable to further explore this new strong promoter due to limitations in time and funding, we hope that our accidental discovery would set out the first step for future explorations.

## **References**

[1] Hou, S., Tong, Y., Yang, H., & Feng, S. (2021). "Molecular Insights into the Copper-Sensitive Operon
Repressor in Acidithiobacillus caldus". Applied and Environmental Microbiology: 87(16).

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## Characterization of csoR-Based System

#### Strains and Growth Medium

E. coli DH10B (taxid: 316385) was used for all routine cloning. LB–Broth medium was used for strain propagation. SOC medium was used for transformation recovery.

#### Plasmids and Genetic Parts

The structure of Transcriptional Unit 1 and Transcriptional Unit 2 is shown in Figure 1. They were designed and synthesized by Ailurus Bio. Single colonies were isolated for further characterization and were Sanger sequenced by 20 bp oligonucleotide (5'­ACGCCCGGTAGTGATCTTAT).

![](https://static.igem.wiki/teams/4494/wiki/exp1.png)

In detail, 100 μl DH10B Chemically Competent Cells (TransGen) were transformed by Transcription Unit 1 and Transcription Unit 2 and then plated on plates containing LB + 2% agar and grown overnight at 37 °C. The plates were stored at 4 °C after growth.

#### mApple Fluorescent Protein Assay

3 single colonies were inoculated into 0.75 ml LB medium with 30 ng/μl chloramphenicol and 50ng/ul Spectinomycin for plasmid maintenance in 2­ml 96­ well deep­well plates (NEST). The plates were sealed with breathable Nunc Seals (Thermo Scientific) and incubated at 37 °C and 1000 r.p.m. overnight. The overnight cultures were diluted 1:325 into 750 μl LB medium with antibiotics in 2­ml 96­well deep­well plates (NEST); the plates were sealed with Nunc Seals and incubated at 37 °C and 1,000 r.p.m. After 3 h, Metal ions were then added at the following concentrations : 0 uM Cu(II), 0.5 uM Cu(II), 5 uM Cu(II), 50 uM Cu(II), 0.5 mM Cu(II), 5 mM Cu(II), 50 mM Cu(II). After mixing by pipetting, 100 μl cultures of each were transferred into 3 standard 96­well plates respectively to set up replicates, sealed with Nunc Seals, and incubated at 37 °C and 1,000 r.p.m. for 16 h. Black 96­Well Cell Culture Plates with Clear Bottom (In Vitro Scientific) were filled with a mixture of 10 μl overnight cultures and 90 μl PBS (Sangon Biotech). The optical density at 600 nm (OD600) and fluorescence readings (560 nm excitation wavelength and 605nm emission wavelength) were carried out on a Spark® Multimode Microplate Reader. Background signals of OD600 and fluorescence, measured from a clear medium, were subtracted from each measurement. Samples with OD600 insignificantly larger than the background were removed to eliminate operational error. mApple per cell was then calculated by dividing the mean fluorescence (A.U.) over the corresponding mean OD600.

## Characterization of CusR-Based System

#### Strains and Growth Medium

E. coli DH10B (taxid: 316385) was used for all routine cloning. LB– Broth medium was used for strain propagation. SOC medium was used for transformation recovery.

#### Plasmids and Genetic Parts

The structure of Transcriptional unit 3 is shown in Figure 2 and was designed and synthesized at Ailurus Bio. Single colonies were isolated for further characterization and Sanger sequenced by 20 bp oligonucleotide (5’­-ATAGGCGTATCACGAGGCAG).

![](https://static.igem.wiki/teams/4494/wiki/exp2.png)

In detail, 100 μl DH10B Chemically Competent Cells (TransGen) were transformed by Transcription Unit 3 and then plated on plates containing LB + 2% agar and grown overnight at 37 °C. Plates were stored at 4 °C after growth.

#### mApple Fluorescent Protein Assay

3 single colonies were inoculated into 0.75 ml LB medium with 50ng/ul Spectinomycin for plasmid maintenance in 2­ml 96­well deep­well plates (NEST). The plates were sealed with breathable Nunc Seals (Thermo Scientific) and incubated at 37 °C and 1000 r.p.m. overnight. The overnight cultures were diluted 1:325 into 750 μl LB medium with antibiotics in 2­ml 96­ well deep­well plates (NEST); the plates were sealed with Nunc Seals and incubated at 37 °C and 1,000 r.p.m. After 3 h, Metal ions were then added at the following concentrations: 0 uM and 50 uM Cu(II). After mixing by pipetting, 100 μl cultures of each were transferred into 3 standard 96­ well plates respectively to set up replicates, sealed with Nunc Seals, and incubated at 37 °C and 1,000 r.p.m. for 16 h. Black 96 ­Well Cell Culture Plates with Clear Bottom (In Vitro Scientific) were filled with a mixture of 10 μl overnight cultures and 90 μl PBS (Sangon Biotech). The optical density at 600 nm (OD600) and fluorescence readings (560 nm excitation wavelength and 605nm emission wavelength) were carried out on a Spark® Multimode Microplate Reader. Background signals of OD600 and fluorescence, measured from a clear medium, were subtracted from each measurement. Samples with OD600 insignificantly larger than the background were removed to eliminate operational error. Protein expression was then calculated by dividing the mean fluorescence (A.U.) over the corresponding mean OD600.

## Engineering and Optimization of csoR-Based System

#### Strains and Growth Medium

E. coli DH10B (taxid: 316385) was used for all routine cloning. LB–Broth medium was used for strain propagation. SOC medium was used for transformation recovery.

#### Plasmids and Genetic Parts

The final structure of Transcription Unit 4 is shown in Figure 3. The synthetic promoter library (Ailurus Bio) and the RBS library were assembled into Transcription Unit 1 (Figure 1) by Golden Gate Assembly at Ailurus Bio. Single colonies were isolated for further characterization and Sanger sequenced by 20 bp oligonucleotide (5’­-ACGCCCGGTAGTGATCTTAT). Notably, the same promoter sequence appeared in different clones.

![](https://static.igem.wiki/teams/4494/wiki/exp3.png)

In detail, the Promoter library and the RBS library were inserted into Transcription Unit 1 sequentially with BsaI­HF®v2 (NEB) and T4 DNA Ligase (NEB) using the program: 37 °C for 5 min, 16 °C for 5 min, 60 cycles. The resulting products were cleaned up using TIANquick Midi Purification Kit (TIANGEN) and then co­transformed with transcriptional unit2 into 100 μl DH10B Chemically Competent Cell (TransGen) and then plated on plates containing LB + 2% agar and grown overnight at 37 °C. Plates were stored at 4 °C after growth.

#### mApple Fluorescent Protein Assay

96 single colonies were inoculated into 0.75 ml LB medium with 30 ng/μl chloramphenicol and 50ng/ul Spectinomycin for plasmid maintenance in a 2­ml 96­ well deep­well plate (NEST), The plates were sealed with breathable Nunc Seals (Thermo Scientific) and incubated at 37 °C and 1000 r.p.m. overnight. The overnight cultures were diluted 1:325 into 750 μl LB medium with antibiotics in 2­ml 96­well deep­well plates (NEST); the plates were sealed with Nunc Seals and incubated at 37 °C and 1,000 r.p.m. After 3 h, Metal ions were then added at the following concentrations: 0 uM Cu(II), 0.5 uM Cu(II), 5 uM Cu(II), 50 uM Cu(II), 0.5 mM Cu(II), 5 mM Cu(II), and 50 mM Cu(II). After mixing by pipetting, 100 μl cultures of each were transferred into 3 standard 96 ­well plates respectively to set up replicates, sealed with Nunc Seals, and incubated at 37 °C and 1,000 r.p.m. for 16 h. Black 96 ­Well Cell Culture Plates with Clear Bottom (In Vitro Scientific) were filled with a mixture of 10 μl overnight cultures and 90 μl PBS (Sangon Biotech). The optical density at 600 nm (OD600) and fluorescence readings (560 nm excitation wavelength and 605nm emission wavelength) were carried out on a Spark® Multimode Microplate Reader. Background signals of OD600 and fluorescence, measured from a clear medium, were subtracted from each measurement. Samples with OD600 insignificantly larger than the background were removed to eliminate operational error. Protein expression was then calculated by dividing the mean fluorescence (A.U.) over the corresponding mean OD600.

## Improvement of BBa\_K1980004

#### Strains and Growth Medium

E. coli DH10B (taxid: 316385) was used for all routine cloning. LB–Broth medium was used for strain propagation. SOC medium was used for transformation recovery.

#### Plasmids and Genetic Parts

The final structure of Transcription Unit 5 is shown in Figure 4. The BBa\_K1980004 mutant library was assembled into Transcription Unit 2 (Figure 2) by Golden Gate Assembly at Ailurus Bio. Single colonies were isolated for further characterization and Sanger sequenced by 20 bp oligonucleotide (5’­-ATAGGCGTATCACGAGGCAG). Notably, the same promoter sequence appeared in different clones.

![](https://static.igem.wiki/teams/4494/wiki/exp4.png)

In detail, the BBa\_K1980004 mutant library was assembled into Transcription Unit 2 sequentially with BsaI­HF®v2 (NEB) and T4 DNA Ligase (NEB) using the program: 37 °C for 5 min, 16 °C for 5 min, 60 cycles. The resulting products were cleaned up using TIANquick Midi Purification Kit (TIANGEN) and then transformed into 100 μl DH10B Chemically Competent Cells (TransGen) and then plated on plates containing LB + 2% agar and grown overnight at 37 °C. Plates were stored at 4 °C after growth.

#### mApple Fluorescent Protein Assay

96 single colonies were inoculated into 0.75 ml LB medium with 50ng/ul Spectinomycin for plasmid maintenance in 2­ml 96-­well deep­well plates (NEST). The plates were sealed with breathable Nunc Seals (Thermo Scientific) and incubated at 37 °C and 1000 r.p.m. overnight. The overnight cultures were diluted 1:325 into 750 μl LB medium with antibiotics in 2­ml 96­well deep­well plates (NEST); the plates were sealed with Nunc Seals and incubated at 37 °C and 1,000 r.p.m. After 3 h, Metal ions were then added at the following concentrations: 0 uM Cu(II) and 50 uM Cu(II). After mixing by pipetting, 100 μl cultures of each were transferred into 3 standard 96 ­well plates respectively to set up replicates, sealed with Nunc Seals, and incubated at 37 °C and 1,000 r.p.m. for 16 h. Black 96­ Well Cell Culture Plates with Clear Bottom (In Vitro Scientific) were filled with a mixture of 10 μl overnight cultures and 90 μl PBS (Sangon Biotech). The optical density at 600 nm (OD600) and fluorescence readings (560 nm excitation wavelength and 605nm emission wavelength) were carried out on a Spark® Multimode microplate Reader. Background signals of OD600 and fluorescence, measured from a clear medium, were subtracted from each measurement. Samples with OD600 insignificantly larger than the background were removed to eliminate operational error. Protein expression was then calculated by dividing the mean fluorescence (A.U.) over the corresponding mean OD600.

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# Experiments

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# Results

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After conducting the experiments presented on our Experiments page, we achieved results and conducted data analysis. The following are the key results.

## Characterization of csoR-Based System

We successfully characterized the csoR-Based System. The system is made out of a combination of Transcription Units 1 and 2 (Figure 1), and the RFU curve is shown in Figure 2. As the RFU kept decreasing, we conclude that the csoR-Based System itself is not sensitive to copper, and that the change in RFU is likely to have been induced solely by the toxicity of copper instead.

![](https://static.igem.wiki/teams/4494/wiki/re1.png)

## Characterization of CusR-Based System

We also characterized the existing CusR-Based System. The system is made out of Transcription Unit 3 (Figure 3), and the RFU curve is shown in Figure 4. We concluded that the currently existing CusR-Based System works as expected, and set it as a benchmark to exceed for our system.

![](https://static.igem.wiki/teams/4494/wiki/re2.png)

## Engineering and Optimization of csoR-Based System

We attempted to optimize the csoR-Based System and produced 96 mutants (78 unique sequences, registered BBa\_K4494003-K4494098). Figure 5 shows the structure of Transcriptional Unit 4, which is used for this experiment. Figures 6 and 7 are respectively the heatmap and scatter plot representations of our results. For more information regarding the experimental results, please visit our [Part Collections](https://2022.igem.wiki/dku-china/part-collection) page.

![](https://static.igem.wiki/teams/4494/wiki/re3.png)

## Improvement of BBa\_K1980004

We tried improving part BBa\_K1980004 to obtain better results for detecting copper and examined its mutants with Transcription Unit 5. We eventually achieved 4 different sequences (registered BBa\_K4494099-K4494102), whose characterizations are respectively shown in Figures 9, 10, 11, and 12. Although our attempts to find a better copper-sensitive promoter have failed, we successfully found a very strong promoter (Figure 12, registered BBa\_K4494102). For more information, please refer to our [Improve](https://2022.igem.wiki/dku-china/improve) page.

![](https://static.igem.wiki/teams/4494/wiki/re4.png)

![](https://static.igem.wiki/teams/4494/wiki/re5.png)

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