iGEM 2022 — Team guangxi-u-china

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# Notebook

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![](https://static.igem.wiki/teams/4157/wiki/plasmid.png)

# Notebook

* [2021](#div1)
* [2022](#div2)

2021

November,2021

| Date | Important Events |
| --- | --- |
| In Nov. of 2021, we sent out the poster for the recruitment of team members to all colleges of Guangxi University. By scanning the WeChat, students who are interested in the iGEM can fill in forms. We have received more than 40 applications from students majored in biology, medicine, electronic engineering, computer science and technology, mechanistic engineering, animal science, economics, journalism and communications. | ![](https://static.igem.wiki/teams/4157/wiki/haibao.png) ![](https://static.igem.wiki/teams/4157/wiki/sign.png) |

November,2021

| Date | Important Events |
| --- | --- |
| Dec. 4 of 2021, we had interviews with the students who meet our requirement. | ![](https://static.igem.wiki/teams/4157/wiki/activity2.png) |
| Dec. 12, 2021, team members were decided. Future plans were discussed. Responsibilities of every team members were preliminarily determined. | ![](https://static.igem.wiki/teams/4157/wiki/activity3.png) |

2022

January, 2022

| Date | Important Events |
| --- | --- |
| Jan. 19 of 2022 | Before winter holiday, 11 articles about breast cancer, immune therapy, tumor microenvironment and glycolysis were distributed to team members. There were 11 team members who decided to make a presentation in February of 2022 to the team. The overall framework was established. |
| After January | Holidays |

February, 2022

| Date | Important Events |
| --- | --- |
| Feb. 15 of 2022 | We had an online Tencent Meeting with medical doctor Lin Zhang who is the chief medical doctor of Tongji Hospital of Huazhong University of Science and Technology, Wuhan, China.  ![](https://static.igem.wiki/teams/4157/wiki/acticity4.png) |

| Date | Important Events |
| --- | --- |
| Feb. 27, 2022 | We had the first brainstorming team meeting. It was an online Tencent Meeting.Yuqi Liu, Luping Zhu, Te Wang, Weixin Cai, Jiangtao Li, Minghao Tang, Yixuan Zhan, Yuanyi Shu, Jiarui Chen, Qian Zhang, Xinyu Wang gave presentation on the literatures distributed in January of 2021.  ![](https://static.igem.wiki/teams/4157/wiki/activity5.png) |

March, 2022

| Date | Important Events |
| --- | --- |
| March 3, 2022 | ![](https://static.igem.wiki/teams/4157/wiki/icon1.png) ![](https://static.igem.wiki/teams/4157/wiki/icon2.png) Our public health WeChat account was launched. |

| Date | Important Events |
| --- | --- |
| March 8, 2022 international Women’s Day. | Our team gave the first 3 public online health lecture that was open to the public. The lectures were talking about the fundamental knowledges of breast cancer for early detection and prevention. Primary PI Professor Hongying Zhong, student members Yuqi Liu and Xinyu Wang gave the presentations.  ![](https://static.igem.wiki/teams/4157/wiki/haibao3.png) ![](https://static.igem.wiki/teams/4157/wiki/haibao4.png) |

| Date | Important Events |
| --- | --- |
| April 2, 2022 | The team had the first non-online meeting of 2022. All team members and candidate members gathered in the lab and discussed the design of the project. Training of fundamentals of synthetic biology and lab safety. |
| April 23, 2022 | Training of synthetic biology and lab safety |

May, 2022

| Date | Important Events |
| --- | --- |
| May2, 2022 | Brainstorming team meeting for the discussion on heterogenous metabolism of glucose and mathematic modeling.   ![](https://static.igem.wiki/teams/4157/wiki/haibao6.png) |
| May 8, 2022 (Mother’s Day) | Online public health education lectures by Tencent Meeting   ![](https://static.igem.wiki/teams/4157/wiki/haibao7.png) |
| May 22, 2022 | Brainstorming team meeting about roles of lactic acid in tumor progression and mathematic modeling. ![](https://static.igem.wiki/teams/4157/wiki/haibao8.png) |

June, 2022

| Date | Important Events |
| --- | --- |
| June 19, 2022 | Team gathering for the clarification of each member’s responsibilities; Trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/activity6.png) |
| June 24, 2022 | Online Tencent Meeting with Professor Ye Yuan of Huazhong University of Science and Technology, Wuhan, China. Professor Ye Yuan is a distinguished professor in AI. ![](https://static.igem.wiki/teams/4157/wiki/haibao9.png) |

July ,2022

| Date | Important Events |
| --- | --- |
| July 2,2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/7-2.jpg) |
| July 9,2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/7-8.jpg) |
| July 17,2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/7-17.jpg) |
| July 23,2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/7-23.jpg) |
| July 30,2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/7-30.jpg) |

August ,2022

| Date | Important Events |
| --- | --- |
| August 6,2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/8-6.jpg) || August 12,2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/8-12.jpg) || August 20,2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/8-20.jpg) |
| August 22,2022 | Public lecture in Fujian ![](https://static.igem.wiki/teams/4157/wiki/hb8-22.jpg) |
| August 24,2022 | Public lecture in Hunan ![](https://static.igem.wiki/teams/4157/wiki/hb8-23.jpg) |
| August 30,2022 | Online Tencent Meeting with professor Bifeng Yuan. He is a distinguished professor in school of public health of Wuhan University.  ![](https://static.igem.wiki/teams/4157/wiki/benyuan.jpg) |
| August 31,2022 | Brainstorming team meeting and trouble shooting. Public lecture in Sichuan and Anhui  ![](https://static.igem.wiki/teams/4157/wiki/8-31.jpg) ![](https://static.igem.wiki/teams/4157/wiki/hb8-31mon.jpg) ![](https://static.igem.wiki/teams/4157/wiki/hb8-31aft.jpg) |
| August 18,2022 | Online meeting with Dr. Brian S DeDecker about the collaboration. |
| August 29,2022 | Online meeting with Dr. Brian S DeDecker about the collaboration. |

September ,2022

| Date | Important Events |
| --- | --- |
| September 1, 2022 | Public lecture in Guangxi ![](https://static.igem.wiki/teams/4157/wiki/hb9-1.jpg) |
| September 3, 2022 | Clarification every member’s job and experimental trouble shooting |
| September 17, 2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/9-17.jpg) |
| September 18, 2022 | Health education and scientific lab show to freshmen ![](https://static.igem.wiki/teams/4157/wiki/9-18.png) |
| September 24, 2022 | Brainstorming team meeting and trouble shooting. ![](https://static.igem.wiki/teams/4157/wiki/9-24.jpg) |

October, 2022

| Date | Important Events |
| --- | --- |
| September 30 to October 12 | Summary |

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# Engineering success

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![](https://static.igem.wiki/teams/4157/wiki/plasmid.png)

# Engineering success

* [Overview](#div1)
* [Genes and primers](#div2)
* [RNases activity and determination](#div3)
* [Co-culture of human cancer cells with E. coli Nissle 1917](#div4)

Overview

In this year, Guangxi-U-China has conducted the engineering of 3 parts including scFv-Fc, SIRPαD1-Fc and acid inducible P-asr promoter for the designed “TuTaBa” bacteria. It is such a joyful journey in which we continuously discover new things and obtain better understanding of the world. The three engineered parts are designed for specific targeting, phagocytic promotion, and acid inducible expression of intended functional genes. Two problems have been solved and two experimental protocols have been established. We have completed the extraction of full-length RNA from breast cancer tumor tissues, reverse transcriptase-polymerase chain reaction (RT-PCR), generation of cDNA for gene cloning, construction of recombinant plasmid and translation in E. coli. Nissle 1917. Their full functions will be characterized in 2023.

Genes and primers

P-atp2

AGTGTCCGTGCTGGGAAACTGGCGGGGAACTTTTAGAGATAACCCTCCGAATTGCTGGCAAATTTCTGATGAAATTTCTCCGCGAAGCCCACATGAACTACCCCCGTTTACCCTCAAAATAAGCCCTGTGACACACATAACACCCCCTAATCGTACCCGCTCACACGCTATTTTCGAGGTGTGGTTCGCCTTCGGAAACGAATGCCCCCGCCCCACTTGGATAAAAGACGAATTCACCTGTTAGTCTATAACGCGGGTTGAACCGAGAAACCCCTCAAGGCAGCAGACAATAGCCGCAAGGGGTTTTGCGGAGCACGTCCCCTGTGATCGTTGCGCTGATGTGCGACGGAGTCCGG

P-asr

CGATCAAGACTACTATTATTGGTAGCTAAATTTCCCTTAAGTCACAATACGTTATTATCAACGCTGTAATTTATTCAGCGTTTGTACATATCGTTACACGCTGAAACCAACCACTCACGGAAGTCTGCCATTCCCAGGGATATAGTTATTTCAACGGCCCCGCAGTGGGGTTAAATGA

IgG1-Fc

AGGTTTGGCGAGTACATAGACCTCTGTTCCCCCTCCAGATTGTATTTCTGTGTCAGGCTCTGATGATCCTTTCTGGAACTTCACACAGTAGTAGATGCCAGCATCTGCTGGGGTGACATTACTGATACGGATGGAAAAGTCCATATTGTTTCTCTTAGTAGTATCTGAAACATTTCTAATTCGAGGAACGTATTCTCCTGCGAAACTGTAGATCAACAGCCGGCTTGGCCCTACTCCTCTGTACCACCTAATGGGTCCCACCGGCAACAAGGAGGTCAAAGTGCAGTTCAGAACGGTCGAATCCCCAGCAGCAACAGACACTGATTTCTCAGGCTGAGTCACCTTCAGTTCCTTCGGATCCG

SIRPaD1

Catttaccaggaggctccttgggaggtggaatggtgtacacctgtggagccttcggtctgcctttggt
tttggagatggttttctcgatgggggcagggaaagctgcactgttgaccctgcatttgaactccttgccattgagccagtcctggtgcatgatgggaagttcactgactgagcggaaagtgctgttgaactgctcctcccggggttgcgtctgagctgtgtgcaagtgggagaggctcttctcagtatggtggttgtgcaggccctcatgtaacacagagcaggtgaaagtatttcctgcctcccagttgctcttctgcacattgagcttgctgtagacgaagtaagagccatttgtgttcatgatgggctgagtgttcttgtagttctccgctggctgcccattccactgccactccacagtaatgtcttcagggaagaagtctgttatcatgcaggtcagactgactttatccttggccatctcctccacatcatctacaaaccagctgaactggacctcgggatcatccttgctgatgtctaccacaacacacgtgaccttaggagtcagagtaatggtgagcacatccttgggctttggggggaagatgaagacagatgatacttctgggactgtacatatgcaaggcttacaaccacaatccctgggcac

anti-CSPG4

ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTACAGTGCTCTAGACAGGTTCAGCTGGTGCAGTCTGGCAGCGAGCTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTAGCGGCTACACATTCACCGACTACAGCATGCACTGGGTCCGAAAGGCCCCTGGACAGGGACTTGAATGGATGGGCTGGATCAACACCGCCACAGGCGAGCCTACCTACGCCGATGGCTTCACAGGCAGATTCGTGATCAGCCTGGACACCAGCGCCAGAACCGTGTACCTGCAGATCAGCTCTCTGAAGGCCGAGGATACCGCCGTGTACTTCTGCTTCAGCTACTACGACTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGACATCGTGCTGACACAGAGCCCTCTGAGCCTGCCTGTTACACCTGGCGAGCCTGCCAGCATCAGCTGTAGAGCCAGCCAGACCATCTACAAGAACCTGCACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATTAAGTACGGCAGCGACAGCATCTCCGGCGTGCCAGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTACACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCTACAGCACCCCTTGGACATTCGGCCAGGGCACCAAGCTGGAAATCAAG

| Table 1. Primers used in gene cloning |  |
| --- | --- |
| Name | Sequence |
| F-EagI- SIRPaD1 | CGAGCTCCTCATTTACCAGGAGAG |
| R-BamHI-SIRPaD1 | AAACCTGTGCCCAGGGATTGTGG |
| F-IgG1(Fc) | GGGCACAGGTTTGGCGAGTACATAG |
| R-IgG1(Fc) | CGGGATCCGAAGGAACTGAAGGTG |
| F-P-asr | CGGGATCCGCGATCAAGACTAC |
| R-p-asr | CCCAAGCTTGTCATTTAACCCCACTG |
| F-P-atp2-HinDIII | AAGCTTAGTGCCGTGCTGGGAAACT |
| F-P-atp2-BamH | GGATCCCGGACTCCGTCGACATCAG |
| F-pET-32b-T7 | TAATACGACTCACTATAGGG |
| R-pET-32b-T7 | GCTAGTTATTGCAGCGG |

RNases activity and determination

Female nude model mice BALB/c that carry with transplanted MCF7 or 4T1 mammary carcinomas have been used for the extraction of full-length RNA samples. Then resultant RNA samples are further subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and generate cDNA for gene cloning. We have experienced difficulties in isolating RNA from BALB/c mice with general experimental protocols. It seems there are strong activities of enzymes that can destroy RNA. The underlying mechanism and roles of RNases in normal and metastatic triple breast cancer have been investigated. We have conducted literature investigation on the functions of RNases and the determination of activities in tumors. Early in 1979, Peterson6 reported serum RNase in the diagnosis of pancreatic carcinoma. Figure 2 illustrates the electrophoretic images of RNA with regular and optimized experimental protocol. The coordination of negative phosphate group in physiological condition with Ti4+ions and the protection of RNA from the degradation has been explored. Figure 3 shows the electrophoretic image of obtained DNA and cloned bacteria.

![](https://static.igem.wiki/teams/4157/wiki/parts-2.png)

Figure 2. Gel electrophoretic imaging of extracted RNA with regular(A) and optimized protocol (B).

![](https://static.igem.wiki/teams/4157/wiki/parts-new.png)

Figure 3. The electrophoretic image of obtained DNA and engineered bacteria.

Co-culture of human cancer cells with E. coli Nissle 1917

The co-culture of epithelial cells and bacteria for investigating host–pathogen interactions was reported in 2009.7 We have established a co-culture protocol of bacteria and breast epithelial cells that can be used for the screening of the colonization and interaction of bacteria to cancer cells. Figure 4 (A)-(C) represent optical images of MDA-MB-231, MCF 7 and MCF 10A that were cultured separately and their co-culture with E. coli Nissle 1917 (non-engineered) were shown in Figure 5 (A)-(C), respectively.

![](https://static.igem.wiki/teams/4157/wiki/parts5.png)

Figure 4. Optical images of MDA-MB-231 (A), MCF 7 (B) and MCF 10A (C).

![](https://static.igem.wiki/teams/4157/wiki/parts6.png)

Figure 5. Optical images of MDA-MB-231 (A), MCF 7 (B) and MCF 10A (C) with E. coli. Nissle 1917 (non-engineered).

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# Measurement and results

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![](https://static.igem.wiki/teams/4157/wiki/plasmid.png)

# Measurement and results

* [Engineering bacteria](#div1)
* [Enzyme activity](#div2)
* [Glucose and lactate](#div3)
* [Metabolome](#div4)
* [Proteome](#div5)

Engineering bacteria

In this year, Guangxi-U-China has conducted the engineering of 3 parts including scFv-Fc, SIRPαD1-Fc and acid inducible P-asr promoter for the designed “TuTaBa” bacteria. The three engineered parts are designed for specific targeting, phagocytic promotion, and acid inducible expression of intended functional genes. We have conducted the extraction of full-length RNA from breast cancer tumor tissues, reverse transcriptase-polymerase chain reaction (RT-PCR), generation of cDNA for gene cloning, construction of recombinant plasmid and translation in E. coli. Nissle 1917. Their full functions will be characterized in 2023.

Electrophoresis of extracted RNA

Female nude model mice BALB/c that carry with transplanted 4T1 mammary carcinomas have been used for the extraction of full-length RNA samples. Figure 1 shows the electrophoretic images of extracted RNA with the optimized experimental protocols.

![](https://static.igem.wiki/teams/4157/wiki/parts-2.png)

Figure 1. Gel electrophoretic imaging of extracted RNA with regular(A) and optimized protocol (B).

Electrophoresis of prepared DNA

Then resultant RNA samples are further subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and generate cDNA for gene cloning. Figure 2 shows the electrophoretic image of cloned DNA and engineered bacteria.

![](https://static.igem.wiki/teams/4157/wiki/parts-new.png)

Figure 2. Gel electrophoretic imaging of DNA and engineered bacteria from the 4th DNA.

Construction of recombinant plasmid and transformation in E. coli. Nissle 1917

Gel pieces containing intended DNA were excised and subjected to further experiments for the recovery of DNA from gel slices with a gel dissolution method. Then conduct a DNA ligation to fuse the insert to the recipient plasmid. Competent cells DH5alpha are used for the transformation. Figure 2 shows that engineered bacteria were only obtained from the 4th band DNA at this time.

Enzyme activity

We have demonstrated activities of RNases are different in normal and tumor tissues. Determination of RNases activities are important not only for the extraction of full-length RNA but also for the understanding of the biological roles of RNases.

Conventional methods involve the extractions of RNases from tissues by which the spatial distribution of enzymatic activities cannot be obtained. An in situ mass spectrometric imaging is established to visualize the activities of RNases in different regions of the tissue. Normal and cancerous tissues are sliced at 10 μm with a freezing microtome. Then either a piece of synthesized marker or endogenous small fragments of RNA can be used as references. Tissue slices are uniformly sprayed and covered with a solution of DHB (2, 4-dihydroxybenzoic Acid). A Nd3+:YAG pulsed laser beam (355 nm) scans across the tissue slice with a pixel size of 20 μm. DHB is used as a matrix material that can absorb laser energy, facilitate soft ionization and protect samples from direct laser ablation. RNases present in tissues cause the degradation of intact references into fragments. Detected intensities of the fragments of references are related with the activities of RNases. Figure 3 shows the mass spectrometric imaging of detected fragments in normal and cancerous tissue slices. It is shown there are heterogenous distribution of detected fragments GGA, GGG and CAG in tissue slices of both BALB/c 4T1 and BALB/c mice models.

![](https://static.igem.wiki/teams/4157/wiki/rnase.png)

Figure 3. Mass spectrometric images of three fragments in different tissue slices.

In summary, we developed a new method for the visualization of activities of RNases based on the detection of small degraded fragments. However, detected fragments of RNA may result from not only in vivo RNases but also in vitro degradation.

Glucose and lactate

A high-performance liquid chromatographic (HPLC) method was developed in this project for the monitoring the dynamics of glucose and lactate metabolism in three different cell lines including MCF 10A, MCF7 and MDA-MB-231. These cells were treated with 3 inhibitors of key regulators glucose transporters, lactate transporters and lactate dehydrogenase. Samples have been collected at different growth time under the treatment with different inhibitors. Because the culture medium contains lots of chemicals, samples were loaded on a reversed phase HPLC C18 column and eluted with a mobile phase containing 30% acetonitrile and 70% water. The pH of the mobile was adjusted as 2 with HCl. Figure 4 shows the chemical structures of glucose and lactate. The detector was set at 210 nm and 285 nm for lactate and glucose, respectively.

![](https://static.igem.wiki/teams/4157/wiki/figure-4-1.png)

Figure 4. Chemical structures of glucose and lactate

Because glucose does not have functional groups for ultraviolet absorption, it was converted to 5-hydroxymethylfurfural in 4.5 mol/L HCl solution at 100°C for 14 minutes as shown in Figure 5. Figure 6 and Figure 7 show the chromatograms of lactate and converted glucose, respectively.

![](https://static.igem.wiki/teams/4157/wiki/figure-5.png)

Figure 5. Conversion of glucose to 5-hydroxymethylfurfural.

![](https://static.igem.wiki/teams/4157/wiki/figure-6.png)

Figure 6. Chromatogram of lactate that was detected at 210 nm

![](https://static.igem.wiki/teams/4157/wiki/figure-7.png)

Figure 7. Chromatogram of glucose that was converted to 5-hydroxymethylfurfural and detected at 285 nm.

A series of standard samples containing known amounts of glucose and lactate was quantified with HPLC. Figure 8 (A) and (B) shows the plots of peak areas vs quantities of lactate and glucose, respectively.

![](https://static.igem.wiki/teams/4157/wiki/figure-8.png)

Figure 8. Plots of peak areas vs quantities of lactate (A) and glucose (B).

In this year, we have mainly set up the analytical procedure. Analysis of samples collected at different growth time under the treatment with different inhibitors have not been completed.

Metabolome

Our project developed a mass spectrometric imaging technique for spatial metabolome analysis of normal and cancerous mice models. It enables the localization of metabolomic changes in different tissue regions. Regular methods are based on the extraction of metabolites from the whole tissues, in which the spatial distribution information is lost. Spatial metabolomics enabled with mass spectrometric imaging (Figure 9) generates big hyper-spectral imaging data that provide rich information on disease progress. Tumor breast tissues (BALB/c 4T1) are sliced as 10 μm in a freezing microtome and then covered with a matrix material called 9-aminoacridine for mass spectrometric imaging in negative ion mode.

![](https://static.igem.wiki/teams/4157/wiki/figure-9.png)

Figure 9. Mass spectrometric imaging with matrix assisted laser desorption ionization (MALDI)

Figure 10 shows the spatial distribution of representative metabolites on a tissue slice that clearly reveal the differences in tumorous and precancerous regions.

![](https://static.igem.wiki/teams/4157/wiki/figure-10.png)

Figure 10. Mass spectrometric imaging of metabolites in a tissue slice from BALB/c 4T1 mouse model.

It shows that saturated fatty acids C16:0, C18:0, C20:0 and monounsaturated fatty acid C18:1are highly abundance in the tumorous region. There are highly heterogenous distribution of metabolites in different regions. In comparison, the spatial distribution of an unknown small molecule at m/z 232.943 is relatively uniform across the whole slice.

Proteome

We have also applied the mass spectrometric imaging technique to the proteomic analysis of normal and tumor tissues of BALB/c mice models. Because the ionization and dissociation of intact proteins are difficult in the mass spectrometer, in situ digestion with trypsin was conducted before the tissue slices were covered with a matrix called α-cyano-4-hydroxycinnamic acid (CHCA) for laser irradiation. Mass spectrometric imaging was performed in the positive ion mode and tryptic peptides are detected. Figure 11 shows the spatial distribution of different peptides on tissue slices of BALB/c and BALB/c 4T1 mice, respectively.

![](https://static.igem.wiki/teams/4157/wiki/r11.png)

Figure 11. The spatial distribution of peptides on tissue slices of BALB/c and BALB/c 4T1 mice.

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