iGEM 2021 — Team NTHU_Taiwan

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# Team:NTHU Taiwan/Notebook

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# Our Notebook

# [NTHUTaiwan](index.html)

* **Give us a call:** +886 03 571 5131
* **Send us a mail:** nthuxigem@gmail.com
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# Team:NTHU Taiwan/Engineering

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* Engineering

######

* [Overview](#section-1)
* [Cloning success](#section-2)
* [Namatode](#section-3)
* [Callus of *Pinus armandii*](#section-4)
* [Dry Lab](#section-5)
* [Design of our model](#section-6)
* [Parameters in this model](#section-7)
* [Iteration](#section-8)
* [Max consumption before pine tree withered](#section-9)

#

# Overview

![](../images/demo/imgr.gif)

For our project, we need to let our plasmid pass through *E.coli*, *Agrobacterium*, pine callus and put *B. xylophilus* on the callus to verify our sequence design. Therefore, we should make sure that each part is on the right track.

For *B. xylophilus*, because we need a large amount of the nematodes for the subsequent experiments, such as qPCR for target genes. We had to maintain their survival without a significant decline in number. So we made every effort to improve the growth environment of *B. xylophilus*.

The subculture of callus needs two weeks, and it is easy to be contaminated by bacteria and influenced by light. It’s better to guarantee both the medium and the process of taking an embryo are not interrupted.

Cloning is the most important part of engineering success. Because our design involves long hairpin RNA which needs to be expressed in pine callus. The choice of proper plasmid and sequence is easier to conduct *Agrobacterium tumefaciens*-mediated gene transfer and hence let the plasmid be able to express on the callus.

To make sure our design is able to kill the nematodes on pine and realize change for nematodes after digesting pine callus with our plasmid, the design for reasonable modeling is also necessary for our project.

To fulfill a scientific design is always not easy; however, we should never give up thinking of the idea to make our project better and complete. The following is the process we improve and reach our goal for each part of our experimental design.

#

# Cloning success

Our experiments were based on previous papers. At the beginning, we wanted to make shRNA expressed in *E.coli*. Since these essays didn’t provide dsRNA sequence, we found some websites which helped us design shRNA. However, after going through Kyoto iGEM 2017 website, we realized that pine wilt nematodes don’t uptake *E.coli*. Therefore, we turned our focus to trees.

We were looking forward to creating transformed pine trees. In our concept, if pine trees could express dsRNA and RNAi mechanisms, this severe issue could be eradicated. Then, we found a handbook which explained the way to conduct hairpin structure cloning on plants. Besides, that book also gave us a novel idea --- long hairpin structure. As a result, we performed cloning experiments according to the handbook. We bought pHANNIBAL plasmids and designed sense and antisense strands. Moreover, we referred to paper to design long hairpin structures --- with whole or 350bp(short version) of exon. Unfortunately, with some unknown contamination, long hairpin construction failed. The sense strand was successfully cloned into pHANNIBAL, but it degraded a few days after the experiment. Thus, we altered our design, using binary vector and shRNA to achieve our goal (Fig. 1-1).

![](https://static.igem.org/mediawiki/2021/b/b0/T--NTHU_taiwan--engineering-1.png)

![](https://static.igem.org/mediawiki/2021/thumb/b/b9/T--NTHU_taiwan--fun3.png/800px-T--NTHU_taiwan--fun3.png)

Substitute P\_r into equation, we have:

Linear:

![](https://static.igem.org/mediawiki/2021/9/9e/T--NTHU_taiwan--fun4.png)

We set [mRNA]\_max as 1, and α/P\_0 can be seen as the toxicity of our system.

Now we have the relation between [mRNA] and the consumption rate. Then, we need to figure out the relation between mRNA and ingestion rate. We consider a cell its cell wall thickness is d and nematode can acquire n nutrient after eating it. Times for a nematode to break down its cell wall is T. Breaking rate for a nematode is proportional to the concentration of pectin lyase p(t), so we could write down:

![](https://static.igem.org/mediawiki/2021/8/83/T--NTHU_taiwan--fun5.png)

Concentration of pectin lyase is dependent on the concentration of mRNA.

![](https://static.igem.org/mediawiki/2021/8/89/T--NTHU_taiwan--fun6.png)
![](https://static.igem.org/mediawiki/2021/d/da/T--NTHU_taiwan--fun7.png)
![](https://static.igem.org/mediawiki/2021/1/11/T--NTHU_taiwan--fun8.png)

Because the ingestion rate of nematode can be written as I=n/T , substitute T from equation (?) into it, we have:

![](https://static.igem.org/mediawiki/2021/0/0d/T--NTHU_taiwan--fun9.png)

The integral rate is proportional to the root of the concentration of mRNA.

#

# Parameters in this model

With these conclusions shown above, we could build a birth-death model and calculate the consumption if we have these parameters: the ingestion rate, the digestion rate, the birth rate, the death rate, and the toxicity.
We could write down the equation of growth for nematodes:

![](https://static.igem.org/mediawiki/2021/4/4e/T--NTHU_taiwan--fun10.png)

The product of A and B is the digestion rate per unit length of nematode. The solution for this equation is:

![](https://static.igem.org/mediawiki/2021/8/89/T--NTHU_taiwan--fun11.png)

Where t\_0 is the hatching time of nematodes, and l\_0 is the length right after the nematodes hatched (initial length).
Fitting the data we found from papers, we have:

![](https://static.igem.org/mediawiki/2021/6/61/T--NTHU_taiwan--dry1.png)

Figure 1: Length of nematode versus time, we choose the hatching point as the zero point of time. Fitting it with l(t)=187.23e^(0.03232(t-23.57))

Fitting result: l(t)=187.23e^(0.03232(t-23.57)). We get the digestion rate.

From the paper, we found the average ages for nematode is roughly 600 hours. To simplify computation, we assume the nematode will die when it aged for 600 hours. We also found that a female nematode can reproduce roughly 80 eggs in 600-74=526 hours. So, we could calculate out the reproduction rate in an hour: 0.076.

As for the initial ingestion rate and the toxicity, we saw them as the control variables. We could compute how much toxicity our modified pine tree needs to prevent withering of the pine trees at a given initial ingestion rate.

| Symbols | Meaning | Description |
| --- | --- | --- |
| l(t) | Length of nematode | Function of times, we can get it by iteration |
| l0 | Length of nematode right after it hatched | 187.23 (mm), we get it from fitting length data |
| D | Digestion rate of nematode | 0.032 (1/hrs), we get it from fitting length data |
| I(t) | Ingestion rate of nematode | Function of times, we can get it by iteration |
| I0 | Initial ingestion rate | Controllable variables |
| B0 | Birth rate of healthy nematodes | 0.076 (1/hrs), we get it from paper |
| [mRNA](t) | Concentration of mRNA | Function of times, we can get it by iteration |
| [mRNA]\_max | Max concentration of mRNA | We set it as 1 because we only need to know the ratio of [mRNA] |
| C(t) | Consumption | Function of time, we can get it by iteration. |
| C\_tot | Total consumption | Accumulation of C(t) |
| P\_rna (C\_tot) | Production rate of mRNA | Function of total consumption, it was discussed in design of model |
| P\_0 | Initial production rate of mRNA |  |
| α | Degradation rate of mRNA induced by consumption |  |
| P | Toxicity of modified pine tree | We defined the ratio of α and P\_0 as the toxicity of the modified pine tree |

Table: Symbols of our model

#

# Iteration

In our model, we use iteration to compute the curve of consumption, birthrate, amount of mRNA, and size. To simplify the computation, we ignore all dimensions except time.

We separate nematodes into 2 kinds to iterate: parent and progeny. Parent nematodes were brought in by the Monochamus alternatus at the beginning of the computation, they were all mature. We thought that the chance for long-horned beetles will be stopped at the same position at the same tree is very small, so the parent nematodes wouldn’t increase in this model. And we set the initial population size of nematodes as 1. The progeny nematodes could be borne by both parent and progeny nematodes.

We mentioned that the consumption rate depends on which is smaller between ingestion rate and digestion rate. The former is the product of the initial ingestion rate and √([mRNA]) , and the latter is the product of digestion rate per length and the length of nematode. If the latter is larger, the consumption rate would decrease, and the birthrate would decrease too. That is, we could write down:

If![](https://static.igem.org/mediawiki/2021/6/67/T--NTHU_taiwan--fun23.png)

![](https://static.igem.org/mediawiki/2021/7/73/T--NTHU_taiwan--fun12.png)
![](https://static.igem.org/mediawiki/2021/3/38/T--NTHU_taiwan--fun19.png)
![](https://static.igem.org/mediawiki/2021/a/ae/T--NTHU_taiwan--fun14.png)
![](https://static.igem.org/mediawiki/2021/b/bf/T--NTHU_taiwan--fun15.png)
![](https://static.igem.org/mediawiki/2021/2/28/T--NTHU_taiwan--fun16.png)

If![](https://static.igem.org/mediawiki/2021/9/93/T--NTHU_taiwan--fun17.png)

![](https://static.igem.org/mediawiki/2021/5/59/T--NTHU_taiwan--fun18.png)
![](https://static.igem.org/mediawiki/2021/3/38/T--NTHU_taiwan--fun19.png)
![](https://static.igem.org/mediawiki/2021/0/0f/T--NTHU_taiwan--fun20.png)
![](https://static.igem.org/mediawiki/2021/b/b5/T--NTHU_taiwan--fun21.png)
![](https://static.igem.org/mediawiki/2021/3/3b/T--NTHU_taiwan--fun22.png)

Bn would be zero for immature nematodes, ln would be constant for mature nematodes.

Here, we only plot the linear mode of the production rate of mRNA, the initial ingestion rate is set to 1. We used red lines to represent parent nematodes and blue lines to represent progeny nematodes. For unmodified pine trees, toxicity is 0. As for modified pine trees, toxicity is 1.

![](https://static.igem.org/mediawiki/2021/b/b8/T--NTHU_taiwan--dry2a.png)

(a)

![](https://static.igem.org/mediawiki/2021/a/ac/T--NTHU_taiwan--dry2b.png)

(b)

![](https://static.igem.org/mediawiki/2021/7/79/T--NTHU_taiwan--dry2c.png)

(c)

![](https://static.igem.org/mediawiki/2021/e/e3/T--NTHU_taiwan--dry2d.png)

(d)

![](https://static.igem.org/mediawiki/2021/7/72/T--NTHU_taiwan--dry2e.png)

(e)

![](https://static.igem.org/mediawiki/2021/5/52/T--NTHU_taiwan--dry2f.png)

(f)

![](https://static.igem.org/mediawiki/2021/2/2f/T--NTHU_taiwan--dry2g.png)

(g)

Figure 2: Iteration result for the unmodified pine trees. (a), (b): birthrate versus time. (c), (d): consumption versus time. (e), (f): [mRNA] versus time. (g): length versus time.

![](https://static.igem.org/mediawiki/2021/5/5a/T--NTHU_taiwan--dry3a.png)

(a)

![](https://static.igem.org/mediawiki/2021/e/e9/T--NTHU_taiwan--dry3b.png)

(b)

![](https://static.igem.org/mediawiki/2021/3/30/T--NTHU_taiwan--dry3c.png)

(c)

![](https://static.igem.org/mediawiki/2021/a/a2/T--NTHU_taiwan--dry3d.png)

(d)

![](https://static.igem.org/mediawiki/2021/5/58/T--NTHU_taiwan--dry3e.png)

(e)

![](https://static.igem.org/mediawiki/2021/3/33/T--NTHU_taiwan--dry3f.png)

(f)

![](https://static.igem.org/mediawiki/2021/2/21/T--NTHU_taiwan--dry3g.png)

(g)

Figure 3: Iteration result for the modified trees. (a), (b): birthrate versus time. (c), (d): consumption versus time. (e), (f): [mRNA] versus time. (g): length versus time.

By iteration result, we could use them to calculate the evolution of population size and the total consumption.

#

# Max consumption before pine tree withered

As mentioned before, the consumption is the main reason why pine trees withered. Therefore, we need to calculate the max consumption in an unmodified pine tree before it withered. According to previous reports, after being infected by nematodes, pine trees would stop secreting resin in 6-20 days. In 20-30 days, the transpiration of pine trees would decline, and leaves would start withering. At the same time, lots of funguses and bacteria invade the sapwood of pine trees. Therefore, nematodes receive lots of nutrients and the population size increases rapidly in the whole tree. We choose 6 days as the point that the consumption is too large for pine trees to cure themselves. We choose 6 days because we want to make sure that all modified pine trees are immune to nematodes. Therefore, we choose the day that pine trees start having symptoms.

![](https://static.igem.org/mediawiki/2021/9/95/T--NTHU_taiwan--dry4a.png)

(a)

![](https://static.igem.org/mediawiki/2021/e/ee/T--NTHU_taiwan--dry4b.png)

(b)

Figure 4: Evolution of population size of nematodes in unmodified pine trees (a) and the accumulation of total consumption (b).

The result is shown above, it shows the evolution of population size and the accumulation of total consumption versus times. And the total consumption in 6 days after being infected is 40.86.

In the same way, we could calculate the evolution of population size and the accumulation of total consumption versus times with any given toxicity and initial ingestion rate. If the population size of nematodes drops to 0.01, we claim that all nematodes are dead in this one tree. By comparing the consumption with the max consumption, we can determine whether this pine tree can survive under the attack of nematodes.

The result of the model is shown in dry lab: model.

# Reference

[1] Xiu-Wen Qiu, Xiao-Qin Wu, Lin Huang, and Jian-Ren Ye. (2016) Influence of Bxpel1 Gene Silencing by dsRNA Interference on the Development and Pathogenicity of the Pine Wood Nematode, *Bursaphelenchus xylophilus*. Int J Mol Sci.17(1): 125.

[2] Han-Yu Fu, Jia-Hong Ren, Lin Huang, Hao Li, and Jian-Ren Ye. (2014) Screening and Functional Analysis of the Peroxiredoxin Specifically Expressed in *Bursaphelenchus xylophilus*—The Causative Agent of Pine Wilt Disease.Int J Mol Sci,15(6): 10215–10232.

[3] Bowen Wang, Xin Hao, Jiayao Xu, Yan Ma, and Ling Ma1. (2019) Transcriptome-Based Analysis Reveals a Crucial Role of BxGPCR17454 in Low Temperature Response of Pine Wood Nematode. Int J Mol Sci, 20(12): 2898.

[4] The Handbook of Plant Functional Genomics: Concepts and Protocols. Chapter 10 RNA Interference. Chris A. Brosnan,Emily J. McCallum,José R. Botella,Bernard J. Carroll. Book Editors:Prof. Dr. Günter Kahl,Prof. Dr. Khalid Meksem.

[5] Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu and Ralph Scorza. (2007) Plum Pox Virus Coat Protein Gene Intron-hairpin-RNA (ihpRNA) Constructs Provide Resistance to Plum Pox Virus in *Nicotiana benthamiana* and *Prunus domestica*. American Society for Horticultural Science Volume 132 issue 6.

[6] Thermo Fisher Scientific BLOCK-iT™ RNAi Designer
https://rnaidesigner.thermofisher.com/rnaiexpress/setOption.do?designOption=shrna&pid=6150814726472832010

[7] Somatic Embryogenesis of Immature Embryos in *Pinus armandii* (accessed Oct 20, 2021).

[8] Liu, X. Z.; Liu, Z.; Yang, Y. M.; Zhang, H. Y. Production of Transgenic *Pinus armandii* Plants Harbouring BtCryIII(A) Gene. Biol. Plant. 2010, 54 (4), 711–714.

[9] Identification and Characterization of a Dual-Acting Antinematodal Agent against the Pinewood Nematode, *Bursaphelenchus xylophilus*, PLoS ONE 4(11):e7593
DOI:10.1371/journal.pone.0007593

[10] 金門國家公園松材線蟲萎凋病防治計畫

[11] https://www.cabi.org/isc/datasheet/10448

# [NTHUTaiwan](index.html)

* **Give us a call:** +886 03 571 5131
* **Send us a mail:** nthuxigem@gmail.com
* **Come visit us:** No. 101, Sec. 2, Guangfu Rd., East Dist., Hsinchu City , Taiwan (R.O.C.) [our location](https://www.google.com/maps/search/300%E6%96%B0%E7%AB%B9%E5%B8%82%E6%9D%B1%E5%8D%80%E5%85%89%E5%BE%A9%E8%B7%AF%E4%BA%8C%E6%AE%B5101%E8%99%9F/%4024.791431%2C120.9915095%2C18z/data%3D%213m1%214b1)

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# Team:NTHU Taiwan/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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# Team:NTHU Taiwan/Results

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* Home
* Wet Lab
* Result

######

* [Cloning](#section-1)
* [Nematode](#section-2)
* [Callus](#section-3)

#

# Cloning

![](../images/demo/imgr.gif)

We used binary vector pBI121 to construct *E. coli* and *A. tumefaciens*. Short-hairpin RNA (shRNA) were designed according to Invitrogen Block-iT RNAi Designer. Therefore, we utilized two primers annealed as target genes aiming to encode shRNA. Since DNA ligation must have phosphate on 5'nucleotides. T4 Polynucleotide Kinase was used to phosphorylate the 5'nucleotides of primers. For plasmid digestion, Sac1 and Xba1 as restriction enzymes, target genes were inserted into pBI121. Besides, we performed gel extraction to prevent self ligation. With CaMV 35S promoter, shRNA could be constitutively expressed in pine trees. Our transformation outcome indicated that shRNA were successfully cloned into plasmid.

![](https://static.igem.org/mediawiki/2021/2/23/T--NTHU_taiwan--f1.png)

# [NTHUTaiwan](index.html)

* **Give us a call:** +886 03 571 5131
* **Send us a mail:** nthuxigem@gmail.com
* **Come visit us:** No. 101, Sec. 2, Guangfu Rd., East Dist., Hsinchu City , Taiwan (R.O.C.) [our location](https://www.google.com/maps/search/300%E6%96%B0%E7%AB%B9%E5%B8%82%E6%9D%B1%E5%8D%80%E5%85%89%E5%BE%A9%E8%B7%AF%E4%BA%8C%E6%AE%B5101%E8%99%9F/%4024.791431%2C120.9915095%2C18z/data%3D%213m1%214b1)

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