iGEM 2024 — Team hk-joint-school

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Wet Lab Notebook

Wet Lab Timeline

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Education Notebook

Education Timeline

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Engineering

* [Inspiration](#des-1)
* [Design](#des-2)
* [Build](#des-3)
* [Test](#des-4)
* [1st Learn](#des-5)
* [1st Improvement](#des-6)
* [2nd Learn](#des-7)
* [2nd Improvement](#des-8)
* [2 engineering cycle (Design-Build-Test-Learn)](#des-9)
* [3rd Learn](#des-10)
* [3rd Improvement](#des-11)
* [References](#des-12)

## **1. Inspiration**

Cancer, especially lung cancer, imposes severe challenges to both the public health and the economy. According to the World Health Organisation, lung cancer is the most prevalent cancer type and accounts for the highest cancer mortality rates in the globe.[1] In general, about 80% to 85% of all lung cancers are Non-Small Cell Lung Cancer (NSCLC). [2] Furthermore, it is predicted that the global economic costs of lung cancer between 2020 and 2050 will amount to $3.9 trillion.[3] In Hong Kong, according to the Hong Kong Cancer Registry, lung cancer accounted for the highest cancer incidence and mortality rate (15.5% and 26.7%). [4] To treat lung cancer, cisplatin, a chemotherapy drug is commonly adopted.[5] However, its clinical use is limited due to severe side effects, ranging from nephrotoxicity, neurotoxicity anototoxicty. These effects are always cumulative and dose-dependent.

After lots of engagement with different stakeholders, we decided to find out ACPs (anticancer peptides) which have combination effects of cisplatin, aiming to reduce toxic effects while enhancing the efficiency of cisplatin.

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## **2. Design**

After some research in the literature, we found that the extracts of *Cordyceps militaris* have anticancer activity on Colorectal cancer and Lung cancer.[6] [7] However, it hasn't been proven exactly which kinds of peptides are effective against lung cancer cell A549. Among the anti-cancer peptides, we have chosen 4 of them to be included in our plasmid first to test their effect. They are CTP-ori, CTP-rds, C-ori and C-rds.[8] By examining ACPred and ENNACT - two distinct databases developed by other researchers – these four peptides shows high score in both database against lung cancer.

The plasmid we designed contains an insert of different ACPs respectively, and ligated with the pET plasmid. The pET plasmid contains kanamycin resistance gene, 6x His tag and a Multiple Cloning Site. In our plasmid design, 2 cut sites, (NdeI and SacI) located before and after SUMO are used to remove the SUMO part and insert the ACPs. NheI is retained for further modification at 5’ end if needed.

![](https://static.igem.wiki/teams/5056/wiki/en/1.png)

###### Fig. 1: initial location of SUMO in pET plasmid

![](https://static.igem.wiki/teams/5056/wiki/en/2.png)

###### Fig. 2: *Cordyceps militaris* peptide insert design

![](https://static.igem.wiki/teams/5056/wiki/en/3.png)

###### Fig. 3: Overview of plasmid design. The insert is C-ori

![](https://static.igem.wiki/teams/5056/wiki/en/4.png)

###### Table 1: Four BioBrick™ of *Cordyceps militaris* peptides designed by our team

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## **3. Build**

All ACP inserts have been incorporated into our pET plasmid and then was tranformed into DH5alpha for cloning. Next, colony PCR would be conducted to check the plasmid. Three out of five pET-ACP plasmids got the positive results (AC-P19, C-ori and C-rds) and were transformed into BL21 to proceed to the next step: protein expression and purification.

During digestion, we encountered a difficulty. As the size of our insert is too small (only 102-132bp), the target band of each inserts are very blur and unclear.

#### *Digestion- 1st attempt*

For each inserts, no target bands are formed after the digestion. One of the possible reasons maybe the size of insert is too short. After discission with our science teachers, we double the concentration of DNA gel stain (SYBR safe DNA strain) from 1X to 2X.

![](https://static.igem.wiki/teams/5056/wiki/en/5.png)

###### Fig. 4: 1st digestion results

#### *Digestion- 2nd attempt*

![](https://static.igem.wiki/teams/5056/wiki/en/6.png)

###### Fig. 5: 2nd digestion results

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## **4. Test**

Concentrations (0, 25 µM, and 50 µM) was investigated (Figure 6). Both peptide drugs C-ori and C-rds did not exhibit significant cytotoxic effects on the cells at the tested concentrations (25 µM and 50 µM). The lack of cytotoxicity also implies that they may not be effective as anticancer agents in this context, as they did not reduce cell viability significantly, even at high concentration as 50 µM.

![](https://static.igem.wiki/teams/5056/wiki/en/7.png)

###### Fig. 6: The effect of peptide drugs C-ori and C-rds on cell viability at different concentrations

After the first experimental engineering cycle and also feedback from different experts, we have gained valuable insights.

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## **5. 1 st Learn**

One of the possible reason why the anticancer effect of C-ori and C-rds to A549 lung cancer cells are weak maybe the score of cell-penetrating activity in CellPPD is low. As it is higher chance that C-ori and C-rds need to enter the cell to function, this may limit its anticancer effect.

![](https://static.igem.wiki/teams/5056/wiki/en/8.png)

###### Table 2: Low scrore of cell pentrating activity in *Cordyceps militaris* anticancer peptides

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## **6. 1 st Improvement**

In order to improve the cell penetrating activity of C-ori and C-rds, a cell-penetrating peptide (CPP) should be added to increase its cell-penetrating activity, thus increasing the anticancer effect. Moreover, a linker is added between the anticancer peptide and CPP. Compared with *Cordyceps militaris* anticancer peptide alone, the fusion protein have higher score of cell-penetrating activity in CellPPD and higher score of anticancer ability in ACPred and ENNACT.

![](https://static.igem.wiki/teams/5056/wiki/en/9.png)

###### Fig. 7: *Cordyceps militaris* peptide + cell-penetrating peptide (CPP) insert design

![](https://static.igem.wiki/teams/5056/wiki/en/10.png)

###### Table 3: Higher score cell pentrating activity in *Cordyceps militaris* fusion anticancer peptides

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## **7. 2 nd Learn**

The mechanism of *Cordyceps militaris* is unknown. As a high school team, based on the limitations of time and resources, it is very difficult for us to figure out the mechanisms of anticancer activity of those peptides, so that we cannot modify the peptides in a right direction easily.

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## **8. 2 nd Improvement**

In order to achieve our goals which is to reduce the side effects of cisplatin by having combination effects with anticancer peptides. We decided to generate some all new peptide (de novo peptides) targeting PDEδ, which regulates KRAS signaling, a common gene mutation in cancerous cells by RFdiffusion and ProteinMPNN-AI.

![](https://static.igem.wiki/teams/5056/wiki/en/11.png)

###### Fig. 8: De novo peptide insert design [BBa\_K5056000- BBa\_K5056004]

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## **2 nd engineering cycle (Design-Build-Test-Learn)**

#### *i) Designing the parts*

According to our results of 1st engineering cycle, we have designed 10 new basic parts.

![](https://static.igem.wiki/teams/5056/wiki/en/12.png)

###### Table 4: Ten new BioBrick™ of anticancer peptides designed by our team

#### *ii) Building the construction*

All ACP inserts have been incorporated into our pET plasmid and then was tranformed into DH5alpha for cloning. Next, colony PCR would be conducted to check the plasmid. Three out of ten pET-ACP plasmids got the positive results (ACP1, ACP5 and C-rds-CPP) and proceed to the next step: protein expression and purification.

#### *iii) Testing the anticancer effect to A549 cell line*

The experimental data showed that the ACPs from *Cordyceps militaris* (C-ori, C-rds, C-rds-CPP) are less effective at killing A549 compared with ACP1 and ACP5 (KAPI). At concentration of 50µM, all peptide drug, except ACP5 (KAPI), has no significant effects compared to the control.

The anti-cancer abilities of ACP5 (KAPI) is much higher than the remaining peptide durgs. At concentration of 50µM, the percentage of cell viability inhibition for A549 with ACP5 (KAPI) is approximately 70%. This shows that ACP5 (KAPI) is the more competent in anti-cancer ability compared with other ACPs.

![](https://static.igem.wiki/teams/5056/wiki/en/13.png)

###### Fig. 9: Cell viability of A549 under different peptide drug treatments at various concentrations

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## **10. 3 rd Learn**

As 6x His-tag is added at the beginning of anticancer peptides, the 6x his-tag may obstract the binding to their target. The size of *Cordyceps militaris* peptides (9 amino acids) and de novo 5 peptide are small (20 amino acids), so it may have a higher chance that 6X his-tag (contains 6 amino acids) may block the binding site / active site of anticancer peptides.

Moreover, the solubility of each anticancer peptides are low. That lowers the production yield and thus increasing the cost.

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## **11. 3 rd Improvement**

We made a new basic part [BBa\_K5056017] which is the modification of BBa\_K5056001. A SUMO-tag and intein are added between 6x his-tag and de novo 5 peptide. The SUMO tag can increase the solubility of the peptides, thus it can increase the production yield. Moreover, intein can help us to remove the 6X-His-tag and SUMO tag by intein self-cleavage. This may enhance the anticancer ability of de novo 5 peptide.

![](https://static.igem.wiki/teams/5056/wiki/en/14.png)

###### Fig. 10: New basic part design [BBa\_K5056017]

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## **References**

1. World Health Organization. (n.d.). (2024, February 1)Global cancer burden growing, amidst mounting need for services. World Health Organization. <https://www.who.int/news/item/01-02-2024-global-cancer-burden-growing--amidst-mounting-need-for-services>
2. What is lung cancer?: Types of lung cancer. Types of Lung Cancer | American Cancer Society. (n.d.).<https://www.cancer.org/cancer/types/lung-cancer/about/what-is.html>
3. Simiao Chen, S. (2023, April 1). Estimates and projections of the global economic cost of 29 cancers from 2020 to 2050. JAMA Oncology.<https://jamanetwork.com/journals/jamaoncology/fullarticle/2801798>
4. Hong Kong Cancer Registry, Hospital Authority. (2023, October). Lung Cancer in 2021. <https://www3.ha.org.hk/cancereg/pdf/factsheet/2021/lung_2021.pdf>
5. American Cancer Society. (n.d.). What is chemotherapy?: Chemo treatment for cancer. What is Chemotherapy? | Chemo Treatment for Cancer | American Cancer Society. <https://www.cancer.org/cancer/managing-cancer/treatment-types/chemotherapy.html>
6. Lee, H. H., Lee, S., Lee, K., Shin, Y. S., Kang, H., & Cho, H. (2015, July 4). Anti-cancer effect of *cordyceps militaris*in human colorectal carcinoma RKO cells via cell cycle arrest and mitochondrial apoptosis. Daru : journal of Faculty of Pharmacy, Tehran University of Medical Sciences. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491205/>
7. Jo, E., Jang, H.-J., Shen, L., Yang, K. E., Jang, M. S., Huh, Y. H., Yoo, H.-S., Park, J., Jang, I. S., & Park, S. J. (2020, May 26). *cordyceps militaris* exerts anticancer effect on non-small cell lung cancer by inhibiting hedgehog signaling via suppression of TCTN3. Integrative cancer therapies. <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265736/>
8. Chantawannakul, J., Chatpattanasiri, P., Wattayagorn, V., Kongsema, M., Noikaew, T., & Chumnanpuen, P. (2021, September 23). Virtual screening for Biomimetic Anti-Cancer peptides from *cordyceps militaris* putative Pepsinized Peptidome and validation on Colon Cancer Cell Line. Molecules (Basel, Switzerland). <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8510206/>

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Experiments

* [Step 1: Plasmid construction](#des-1)
* [Step 2: Transformation](#des-2)
* [Step 3: Colony PCR](#des-3)
* [Step 4: Protein expression](#des-4)
* [Step 5: Purification of AC-P19](#des-5)
* [Step 6: Preliminary cytotoxicity test of AC-P19 for A549 cell line](#des-6)
* [Step 7: Protein expression of  *militaris* peptides](#des-7)
* [Step 8: Purification of *Cordyceps militaris* peptides](#des-8)
* [Step 9: Drug screening of Cisplatin and *Cordyceps militaris* peptides for A549](#des-9)
* [Step 10: Cytotoxicity test for peptides w/CPP](#des-10)
* [Step 11: Cytotoxicity test of KAPI for A549 & BEAS-2B](#des-11)
* [Step 12: Combination effect of KAPI & cisplatin](#des-12)
* [Step 13: Drug screening by 3D spheroid](#des-13)
* [Step 14: qPCR](#des-14)
* [Appendix](#des-15)

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Results

* [Achievements](#des-1)
* [BioBrick™ Assembly](#des-2)
* [Protein production](#des-3)
* [Preliminary cytotoxicity test of AC-P19 for A549 cell line](#des-4)
* [Drug screening of Cisplatin for A549 Cell line](#des-5)
* [Drug screening of *Cordyceps militaris* peptides for A549 Cell line](#des-6)
* [Drug screening of ACP1, ACP5 and C-rds-CPP compared with *Cordyceps militaris* peptides for A549 Cell line](#des-7)
* [Further cytotoxicity test of ACP5 (KAPI) for A549 cell line](#des-8)
* [Further cytotoxicity test of ACP5 (KAPI) for BEAS-2B cell line](#des-9)
* [The combination effect of KAPI and cisplatin](#des-10)
* [Drug screening by 3D spheroid](#des-11)
* [qPCR](#des-12)
* [Future plan](#des-13)
* [Reference](#des-14)

## **1. Achievements**

Our project aims to design and produce an ACP which can have combination effects with cisplatin against Non-Small Cell Lung Cancer (NSCLC). We have total designed fifteen BioBrick™ to express different anticancer peptides in our expression system. AC-P19 is an anticancer peptide which has been experimentally proved that it has anticancer ability against A549 cell line [BBa\_5056009]. [1] We used it as a positive control. Four peptides from *Cordyceps militaris*, a well-known dietary therapy in anticancer treatment, were selected to test their effectiveness to lung cancer [BBa\_5056005]-[BBa\_5056008]. Also, we modified those five peptides (AC-P19, C-ori, C-rds, CTP-ori and CTP-rds) by adding linker and cell-penetrating peptide to increase anticancer ability via promoting cell-penetrating ability [BBa\_5056011]-[BBa\_5056015]. Furthermore, we used RFdiffusion and ProteinMPNN-AI to generate all-new peptide sequences by targeting PDEδ, which regulates KRAS signaling, a common gene mutation in cancerous cells. Linker and cell-penetrating peptide are also added at end of those de novo peptides to increase its cell-penetrating ability [BBa\_5056000]-[BBa\_5056004].

![](https://static.igem.wiki/teams/5056/wiki/more-results/actual-table-1.jpeg)

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