iGEM 2024 — Team hubu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

Contents

## **April 22****th****, 2024** Selection of Target Genes

*Spodoptera litura* is an important crop pest, which has evolved a high degree of resistance to all insecticides targeted at it. After 24 h injection of 500 ng/μL dsRNA, the expression levels of SlCHS1 and SlCHS2 were significantly inhibited in *S. litura* (Yu et al.,2020). Among them, *SlCHS1* has a high expression level in the outer epidermis and is exclusively expressed in the epidermis underlying the cuticular exoskeleton and related ectodermal cells such as tracheal cells, while *SlCHS2* is highly expressed in the midgut, which protects the intestinal epithelium from mechanical disruption and invasion by various pathogens.

These results suggest that *SlCHS1* might play an important role during *S. litura* larval molting, while *SlCHS2* has no significant effect on *S. litura* molting. Therefore, we have chosen *SlCHS1* for further study.

![clip_image001.png](https://static.igem.wiki/teams/5044/clip-image001.png)

**Reference**

Yu HZ, Li NY, Xie YX, et al. (2020) Identification and functional analysis of two chitin synthase genes in the common cutworm, *Spodoptera litura*. Insects 11: 4.

## **April 23****th****, 2024** Selection of amiRNA Backbones

Five backbones were selected in the literature (Bally et al., 2020): At-miR159(*At, Arabidopsis thaliana*), Dm-ba (*Dm, Drosophila melanogaster*), Dv-miR279 (*Dv, Diabrotica virgifera*), Dv-miR1 and Tc-ba (*Tc, Tribolium castaneum*). The literature shows that even if the inhibitory effect of Artificial microRNA (amiRNA) delivered in the *Arabidopsis* pre-miR backbone is lower than that of amiRNA delivered in the insect pre-miR backbones, the effect of the insect backbones is better than that of the plant backbones. The results of the five backbones compared with the wild type are as follows:

![clip_image002.png](https://static.igem.wiki/teams/5044/clip-image002.png)

By comparing the rate of death and pupation failure：greatest inhibition Tc-ba > Dv-miR279 > Dv-miR1 > Dm-ba > At-miR159, so we finally chose the Tc-ba backbone.

**Reference**

Bally J, Mclntyre G J, Doran R L, et al. (2016) In-plant protection against *Helicoverpa armigera* by production of long hpRNA in chloroplasts. Frontiers in Plant Science, 7, 1453.

## **April 24****th****, 2024** Prediction of Effective Fragments of CHS1 from Spodoptera litura

The relative location and number of effective amiRNA candidates at *CHS1* were predicted by indicated bioinformatics tools (DNAMAN, siDirect and Wizard). We selected hotspots predicted by all three tools as effective fragments of *CHS1* from *Spodoptera litura*. Two amiRNA candidates (CHS1-1, CHS1-2) are in the hotspot.

***Spodoptera litura*** *CHS1*

**amiRNA-1:** TGGATGACAACGTCATGAAGAAG

**amiRNA-2:** AAGGTTTCAACGAGTTCTACAAT

## **April 25****th****, 2024** The Construction of Tc-ba-miR-CHS1

We found the base sequence of Tc-ba-miR-ACE2 (Yu et al.2020).

Tc-ba: Tribolium castaneum bantam H. armigera ACE2 target

GAGGAATTCACCTCGTCACGGCGACGTCAGCGAGACGACCATGGAGTTGGATGACAGGAAGGAGATATATTTGATTCTCTCCTGTCATCCAACTCCATGGTTGTTCATTAATCATCGATCGCAGGCAGCACCCCTTAGAA

By replacing *ACE2* with *CHS1* in Tc-ba-miR-ACE2, Tc-ba-miR-CHS1 was constructed.

Tc-ba scaffold S. litura CHS1 target 1

GAGGAATTCACCTCGTCACGGCGACGTCAGCGAGACGACCGATGACAACGTCATGAAGAAGGATATATTTGATTCCTTCTTCATGACGTTGTCATCGGTTGTTCATTAATCATCGATCGCAGGCAGCACCCCTTAGAA

## **April 26****th****, 2024** Primers were Designed and Synthesized by Wuhan Gencreate Company in China

Tc-ba-miR-CHS1-1 synF1

GAGGAATTCACCTCGTCACGGCGACGTCAGCGAGACGACCGATGACAACGTCATGAAGA

Tc-ba-miR-CHS1-1 synF2

TGACAACGTCATGAAGAAGGATATATTTGATTCCTTCTTCATGACGTTGTCATCGGTTG

Tc-ba-miR-CHS1-1 synF3

TTCCGTGCTGCCTGCGATCGATGATTAATGAACAACCGATGACAACGTCATGAA

XhoI-Tc-F1

AAAACTCGAGTTCTAAGGGGTGCTGCCT

XbaI-Tc-R1

AAAATCTAGAACATGAGGATCACCCATGTGAGGAATTCAC

## **May 2****nd****, 2024:** Get the Interest Fragment

**PCR Reaction system**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Tc-ba-miR-CHS1-1 synF1 | 2 μL |
| Tc-ba-miR-CHS1-1 synF3 | 2 μL |
| Golden Star T6 Super PCR Mix (Beijing Tsingke Biotechnology Co., Ltd., China) | 46 μL |
| Total volume | 50 μL |

Place in a PCR amplifier (Adjust the time according to the instructions).

**1.  Agarose gel preparation for electrophoresis**

* Prepare 1 % agarose gel.
* Weigh 0.5 g of agarose into a conical flask and add 50 ml of Tris-acetate EDTA (TAE).
* Heat in the microwave until transparent.
* Select the comb according to the fragment length and insert a comb at the top of the mold to form the sample well.
* After the gel is polymerized, the comb can be removed.
* Place gel in the electrophoresis chamber. Gels should be completely submerged in buffer to allow ion flow and prevent gel drying.

**2.  Electrophoresis**

* Remove the three PCR tubes from the PCR amplifier.
* Load the marker (2k Plus2) into the first well using a micropipette and allow all the samples in the three PCR tubes to “fall” into each of the three the sample.
* Fill the upper and lower chambers with running buffer and apply an electric potential on the gel at a constant voltage of 120 V.
* Remove the gel from the gel tray and expose the gel to UV light. DNA bands show up as fluorescent bands.

![图片1.png](https://static.igem.wiki/teams/5044/1.png)

**3.  Recycling of DNA from Agarose Gels**

* Excise a gel slice containing the DNA of interest.
* Place the gel slice containing the DNA sample into an EP tube. Add 30μl of XP2 Binding Buffer (Omega Bio-Tek, USA).
* Melt the gel slice at 70°C for 15 min. Make sure that the gel slice is melted.
* Pour in the Spin column to collect Tube and centrifugal (12000 rpm 1 min).
* Discard the buffer.
* Add 700μl SPW Buffer (Omega Bio-Tek, USA) to elute 2 times. Centrifuge the solution (12000 rpm 2 min).
* Replace the lower part with an EP tube.
* Add 30μl Elution buffer and heat for 2 min at 60 °C. Centrifuge the solution (12000 rpm 1 min).
* Nucleic acid quantification.
* Get the fragment Primer 2＋3.

## **May 3****rd****, 2024:** Get the Interest Fragment

**1.  Reaction system:**

| Reagent | Volume |
| --- | --- |
| Primer2＋3 | 1 μl |
| Tc-ba-miR-CHS1-1 synF1 | 2 μl |
| Tc-ba-miR-CHS1-1 synF3 | 2 μl |
| Golden Star T6 Super PCR Mix (Beijing Tsingke Biotechnology Co., ltd., China) | 45 μl |
| Total volume | 50 μl |

Place in a PCR amplifier (Adjust the time according to the instructions).

**2.  Prepare with 1 % agarose gelatin.**

**3.  Electrophoresis.**

**4.  Take a picture of the gel.**

![11111.png](https://static.igem.wiki/teams/5044/11111.png)

**5.  Recycling of DNA from Agarose Gels.**

     Get the interest fragment Tc-ba-miR-CHS1.

####

## **May 4****th****, 2024:** Sequence of Digestion Sites Added

**1.  Reaction system:**

| Reagent | Volume |
| --- | --- |
| Tc-ba-miR-CHS1 | 1 μl |
| XhoI-Tc-F1 | 2 μl |
| XbaI-Tc-R1 | 2 μl |
| Golden Star T6 Super PCR Mix (Beijing Tsingke Biotechnology Co., Ltd., China) | 45 μl |
| Total volume | 50 μl |

**2.  Prepare with 1 % agarose gelatin.**

**3.  Electrophoresis.**

**4.  Take a picture of the gel.**

![44444.png](https://static.igem.wiki/teams/5044/44444.png)

**5.  Recycling of DNA from Agarose Gels.**

Get the interest fragment containing digestion sites (XhoI XbaI).

## **May 5****th****, 2024:** Plasmid Transformation

**1.  Plasmid transformation.**

After receiving the plasmid pET-28a (+) dry powder, follow the instructions on the kit to transform the plasmid into E. coli DH5α.

**2.  Culture monoclonal colonies.**

* Add 900 μl of antibiotic-free LB liquid medium and incubate at 180 rpm
        shaking for 45 min.
* Centrifuge at 6000 rpm for 5 min.
* Inoculate onto Luria-Bertani (LB) medium containing Kanamycin. Incubate the culture overnight at 37°C.

![5555.png](https://static.igem.wiki/teams/5044/5555.png)

## **May 6****th****, 2024:** Culture Monoclonal Colonies

Pick monoclonal colonies to inoculate 2 ml of LB medium containing Kan with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.

## **May 7****th****, 2024:** Plasmid Extraction

**1.  Lyse bacteria to release plasmid DNA.**

* Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at 12000 rpm for 30 s at 4 °C.
* Resuspend the bacterial pellet in 100 µl of ice-cold Alkaline lysis solution I by vigorous vortexing.

**2.  Isolate and purify plasmids.**

* Add 200 µl of Alkaline lysis solution II to each bacterial suspension.
* Add 150 µl of ice-cold Alkaline lysis solution III.
* Centrifuge the bacterial lysate at 12000rmp for 5 min at 4 °C in a microfuge. Transfer the supernatant to a fresh tube.
* Add an equal volume of phenol: chloroform.
* Adding ethanol into the supernatant. Mix the solution by vortexing.
* Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 min at 4 °C in a microfuge.

**3.  Measure nucleic acid concentration.**

**4.  Store at -20°C for later use.**

![](https://static.igem.wiki/teams/5044/notebook-5-7.jpg)

## **May 8****th** **,2024:** The Expression Vector pMJ5 Was Designed Based on pET28a (+)

![66666.png](https://static.igem.wiki/teams/5044/66666.png)

## **May 9****th** **,2024:** Plasmid Digestion

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| XbaI | 1 μl |
| XhoI | 1 μl |
| pET-28a（3÷0.2） | 15 μl |
| 10X QuickCut Green Buffer | 5 μl |
| ddH2O | 28 μl |
| Total volume | 50 μl |

2.  37 ℃, 5h.

3.  Agarose gel preparation for electrophoresis.

4.  Electrophoresis.

5.  Recycling of DNA from agarose gels.

![5.png](https://static.igem.wiki/teams/5044/5.png)

6.  Measure nucleic acid concentration.

7.  Store at -20 °C for later use.

## **May 10****th** **,2024:** PCR product Digestion

**1****.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| XbaI | 1 μl |
| XhoI | 1 μl |
| Tc-ba-miR-CHS1 (containing digestion sites) | 30 μl |
| 10X QuickCut Green Buffer | 3 μl |
| ddH2O | 15 μl |
| Total volume | 50 μl |

**2.  37 ℃, 5 h.**

**3.  Agarose gel preparation for electrophoresis.**

**4.  Electrophoresis.**

**5.  Recycling of DNA from agarose gels.**

![图片5.png](https://static.igem.wiki/teams/5044/5.png)

**6.  Measure nucleic acid concentration.**

**7.  Store at -20 °C for later use.**

## **May 11****th** **,2024:**the Plasmid and Fragment are Ligated by the T4 DNA Ligase

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| processed vector | 1 μl |
| processed Tc-ba-miR-CHS1 | 3 μl |
| T4 DNA Ligase | 0.5 μl |
| 10 × T4 DNA ligase Buffer | 1 μl |
| ddH2O | 4.5 μl |
| Total volume | 10 μl |

**2.  16 ℃, 3 h.**

**3.  The constructed plasmid was transformed into DH5α by heat shock.**

* Add 5 μl of the constructed plasmid mixture and place on ice for 20 min.
* Heat shock 42 ℃, 45 s.
* Add 500 μl of LB medium to each of the tubes.
* Shake at 211 rpm, 37 ℃ for 1 h.

**4.  Add 300 μl of the above transformation mix to a solid LB containing Kana (100 μg /ml) in a clean bench.**

**5.  Overnight at 37 °C.**

## **May 12****th** **,2024:** Check whether the Vector is Successfully Constructed

**1.  Colony PCR:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| 10 µM Primer F | 2 µl |
| 10 µM Primer R | 2 µl |
| Golden Mix (green) | 45 µl |
| Bacterial liquid | 1 µl |
| Total volume | 50 µl |

**2.  Agarose gel preparation for electrophoresis.**

**3.  Electrophoresis.**

![图片6.png](https://static.igem.wiki/teams/5044/6.png)

## **May 14****th** **,2024:** Single Colony Expansion Culture

**1.  Pick the sequenced single colonies in the clean bench and add them to a bacterial bottle (contains 5 ml of liquid medium containing KanR).**

**2.  Overnight on a shaker at 37 °C.**

## **May 15****th** **,2024:** Plasmid Extraction

**1.  The bacterial fluid in the bacterial bottle appears cloudy.**

**2.  Plasmid Extraction (Operate according to the instructions).**

**3.  Measure nucleic acid concentration.**

**4.  Take 10 μl for sequencing.**

**5.  The company emailed back that the fragment sequenced yesterday was correct. Then the vector pMJ5 (Tc-ba-miR-CHS1) was constructed successfully.**

## **May 18****th** **,2024:** The Expression Vector pMJ6 Was Designed Based on pFJ1

**1.  The pFJ1 is made by the senior sister and we replaced the target gene on this basis**

![图片8.png](https://static.igem.wiki/teams/5044/8.png) ![图片9.png](https://static.igem.wiki/teams/5044/9.png)

## **May 19****th** **,2024:** Two Primers were Designed and Synthesized by Wuhan Gencreate Company in China

XhoI-Tc-F2

AAAACTCGAGTTCTAAGGGGTGCTGCCTGCGATCG

HindⅢ-Tc-R2

AAAAAAGCTTGAGGAATTCACCTCGTCACGGCGAC

## **May 20****th****, 2024:** Get the Interest Fragment

Received two primers (Primer1: XhoI-Tc-F2 Primer2: HindⅢ-Tc-R2)synthesized by the company.

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Tc-ba-miR-CHS1 | 1 μl |
| XhoI-Tc-F2 | 2 μl |
| HindⅢ-Tc-R2 | 2 μl |
| Golden Star T6 Super PCR Mix (Beijing Tsingke Biotechnology Co.,Ltd., China) | 45 μl |
| Total volume | 50 μl |

**2.  Prepare with 1 % agarose gelatin.**

**3.  Electrophoresis.**

**4.  Take a picture of the gel.**

![图片10.png](https://static.igem.wiki/teams/5044/10.png)

**5.  Recycling of DNA from Agarose Gels.**

        Get the interest fragment containing digestion sites (XhoI HindⅢ).

## **May 21****th** **,2024:** Plasmid Digestion

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| HindⅢ | 1 μl |
| XhoI | 1 μl |
| pJF1（3÷0.2） | 15 μl |
| 10X QuickCut Green Buffer | 5 μl |
| ddH2O | 28 μl |
| Total volume | 50 μl |

**2.  37 ℃, 5h.**

**3.  Agarose gel preparation for electrophoresis.**

**4.  Electrophoresis.**

**5.  Recycling of DNA from agarose gels.**

![图片11.png](https://static.igem.wiki/teams/5044/11.png)

**6.  Measure nucleic acid concentration.**

**7.  Store at -20 °C for later use.**

## **May 22****th** **,2024:** the Plasmid and Fragment are Ligated by the T4 DNA Ligase

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| processed vector | 1 μl |
| processed interest fragment | 2.5 μl |
| T4 DNA Ligase | 0.5 μl |
| 10 × T4 DNA ligase Buffer | 1 μl |
| ddH2O | 5 μl |
| Total volume | 10 μl |

**2.  16 ℃, 3 h.**

**3.  The constructed plasmid was transformed into DH5α by heat shock.**

* Add 5 μl of the constructed plasmid mixture and place on ice for 20 min.
* Heat shock 42 ℃, 45 s.
* Add 500 μl of LB medium to each of the tubes.
* Shake at 211 rpm, 37 ℃ for 1 h.

**4.  Add 300 μl of the above transformation mix to a solid LB containing Kana (100 μg /ml) in a clean bench.**

**5.  Overnight at 37 °C.**

## **May 23****th** **,2024:** Check whether the Vector is Successfully Constructed

**1.  Colony PCR:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| 10 µM Primer F | 2 µl |
| 10 µM Primer R | 2 µl |
| Golden Mix (green) | 45 µl |
| Bacterial liquid | 1 µl |
| Total volume | 50 µl |

**2.  Agarose gel preparation for electrophoresis.**

**3.  Electrophoresis.**

![图片12.png](https://static.igem.wiki/teams/5044/12.png)

## **May 24****th** **,2024:** the Plasmid and Fragment are Ligated by the T4 DNA Ligase

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| processed vector | 1 μl |
| processed interest fragment | 2.5 μl |
| T4 DNA Ligase | 0.5 μl |
| 10 × T4 DNA ligase Buffer | 1 μl |
| ddH2O | 5 μl |
| Total volume | 10 μl |

**2.  16 ℃, 3 h.**

**3.  The constructed plasmid was transformed into DH5α by heat shock.**

* Add 5 μl of the constructed plasmid mixture and place on ice for 20 min.
* Heat shock 42 ℃, 45 s.
* Add 500 μl of LB medium to each of the tubes.
* Shake at 211 rpm, 37 ℃ for 1 h.

**4.  Add 300 μl of the above transformation mix to a solid LB containing Kana (100 μg /ml) in a clean bench.**

**5.  Overnight at 37 °C.**

## **May 25****th** **,2024:** Check whether the Vector is Successfully Constructed

**1.  Colony PCR:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| 10 µM Primer F | 2 µl |
| 10 µM Primer R | 2 µl |
| Golden Mix (green) | 45 µl |
| Bacterial liquid | 1 µl |
| Total volume | 50µl |

**2.  Agarose gel preparation for electrophoresis.**

**3.  Electrophoresis.**

![图片13.png](https://static.igem.wiki/teams/5044/13.png)

## **May 26****th** **,2024:**Single Colony Expansion Culture

**1.  Pick the sequenced single colonies in the clean bench and add them to a bacterial bottle (contains 5 ml of liquid medium containing KanR).**

**2.  Overnight on a shaker at 37 °C.**

## **May 27****th** **,2024:** Plasmid Extraction

**1.  The bacterial fluid in the bacterial bottle appears cloudy.**

**2.  Plasmid Extraction (Operate according to the instructions).**

**3.  Measure nucleic acid concentration.**

**4.  Take 1 μg plasmid for sequencing.**

**5.  The company emailed back that the fragment sequenced yesterday was correct. Then the vector pMJ6 (MS2-Tc-ba-miR-CHS1) was constructed successfully.**

![图片14.png](https://static.igem.wiki/teams/5044/14.png)

## **June 7****th** **,2024:** E. coli B2 (BL21ΔRNC) were Prepared as Competent

**1.  Melt the E. coli BL21 strain stored at -80 °C on ice.**

**2.  Plate streak on LB medium.**

**3.  Overnight at 37 °C.**

## **June 8****th** **,2024:** E. coli B2 (BL21ΔRNC) were Prepared as Competent

**1.  Pick BL21 single colonies from LB medium and added to 100 mL of LB liquid medium.**

**2.  Shake at 200 rpm, 37 °C for 48h.**

![](https://static.igem.wiki/teams/5044/notebook-6-8.jpg)

## **June 9****th** **,2024:** E. coli B2 (BL21ΔRNC) were Prepared as Competent

**1.  When the bacterial solution OD600 nm=0.4~0.5, place it on ice for 30min**

**2.  Add the bacterial solution to 50ml of liquid medium and put it on ice for 10 min**

**3.  4℃、Centrifuge (3 000 rpm 5 min), remove the liquid from the tube**

**4.  Resuspend the cells with 20 ml of pre-chilled CaCl2, and put it on ice for 10 min**

**5.  Repeat the above two steps**

**6.  After aliquoting, it is quickly frozen with liquid nitrogen, store at -80 °C for later use**

![](https://static.igem.wiki/teams/5044/notebook-6-9.png)

## **June 10****th** **,2024:** Plasmid Transformation

**1.  Three plasmids (pMJ5 pMJ6 pMJ7) were transformed into competent cells of B2 (BL21ΔRNC) at room temperature.**

* Take the competent prepared yesterday and melt it on ice
* Take 30 μl of competent and add it to the tube containing the plasmid, put on ice for 30min
* Place in water at 42 °C for 45 s, then immediately place on ice for 2min
* Add to 500 μl of medium and shake at 200 rpm, 37 °C for 1 h

**2.  Add 300 μl of the above transformation mix to a solid LB containing Kana (100 μg /ml) in a clean bench.**

**3.  Overnight at 37 °C.**

## **June 11****th** **,2024:** Check whether the Vector is Successfully Constructed

**1.  Colony PCR:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| 10 µM Primer F | 2 µl |
| 10 µM Primer R | 2 µl |
| Golden Mix (green) | 45 µl |
| Bacterial liquid | 1 µl |
| Total volume | 50 µl |

**2.  Agarose gel preparation for electrophoresis.**

**3.  Electrophoresis.**

![](https://static.igem.wiki/teams/5044/nootbook-6-11.png)

**4.  Pick the 3 colonies with the brightest bands and add them to 100 ml of liquid medium. The same procedure is followed for bacteria containing pFJ4 and pET28a. Shake at 200 rpm, 37 °C overnight**

## **June 11****th** **,2024:** Check whether the Vector is Successfully Constructed

**1.  When the bacterial solution OD600 nm=0.4~0.5, place it on ice for 30min**

**2.  Plasmid Extraction (Operate according to the instructions).**

**3.  Measure nucleic acid concentration.**

**4.  Take 10 μl for sequencing.**

**5.  The company emailed back that the fragment sequenced yesterday was correct. Then the five vector were constructed successfully.**

## **June 12****th** **,2024:** Expanded Culture

**1.  Each of the 5 bacteria was added to 1 L of LB liquid medium (1mmol/L).**

**2.  Shake at 200 rpm, 37 °C overnight.**

![](https://static.igem.wiki/teams/5044/notebook-6-12.png)

## **June 13****th** **,2024:** Induced Expression

**1.  When the bacterial solution OD600 nm=0.6~0.8, place it on ice for 30min**

**2.  Inoculate LB medium ( IPTG 1 mM/L) and overnight at 37℃**

## **June 14****th** **,2024:** The RNA expression levels were evaluated using Northern blot analys

**1.  Extract total RNA from the samples using TRIzol reagent.**

**2.  Treat the RNA samples with formaldehyde and heat to denature them for analysis by gel electrophoresis.**

![notebook 6.14.png](https://static.igem.wiki/teams/5044/notebook-6-14.png)

Northern blot results showed that amiRNA expression was detected in the B2 strains expressing pMJ5, pMJ6 and pMJ7 (138 nt)

## **June 15****th****,2024:** SDS-PAGE was Used to Detect whether the Protein was Expressed

**1.  Protein extraction**

**2.  Glue pouring and sample loading**

**3.  Gel electrophoresis**

**4.  Dyeing and decolorization**

![notebook 6.15.png](https://static.igem.wiki/teams/5044/notebook-6-15.png)

      The results of SDS-PAGE assay showed that the expression of 2× CP (28 kDa) was detected in the strains expressing pFJ4 and pMJ6, and the expression of GFP (29 kDa) was detected in the strains expressing pMJ7

**5.  The expression strain was constructed**

## **June 18****th****,2024**

1.  Preparation of fungus

The plasmid was transferred into five kinds of B2 bacterial solution, and after thawing, it was inoculated into 10mL liquid LB medium containing 50mg/L Kana at 1% inoculation rate, and then oscillated and activated in a shaking table at 37℃ overnight. When OD600=0.6, the activated bacterial solution was absorbed into 1L liquid LB medium containing 50mg/L Kana (kanamycin) at 1% inoculation rate, and incubated in a shaking table at 37℃ for 6h. When OD600=0.6-0.8 was measured, 1mL 1mol/L IPTG was added to the mixture (stored in the refrigerator at -20℃), and continued to shake and culture, and induced overnight. The bacteria were collected by centrifuge at 6000rpm for 5min in a large-sized floor-mounted high-speed refrigerated centrifuge. After adding Tris-HCl 8.0, the bacteria were shaken and mixed, and stored at −80℃ for future use.

![notebook 6.18.png](https://static.igem.wiki/teams/5044/notebook-6-18.png)

## **June 19****th****,2024**

1.  Feed preparation

Prepare 25 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

A total of 250 larvaes were placed in small feed boxes (10 larvaes of Spodoptera litura in each box).

## **June 20****th****,2024**

1.  Feed preparation

Prepare 25 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for 250 larvaes of Spodoptera litura (10 larvae of Spodoptera litura in each box).

## **June 21****th****,2024**

1.  Feed preparation

Prepare 25 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.   Biological Detection

Replacement of feed box for 250 larvaes of Spodoptera litura (10 larvae of Spodoptera litura in each box).

3.  Observe, record the number of surviving Spodoptera litura. Remove 5 Spodoptera litura from 50 heads of surviving Spodoptera litura for weight weighing, remove 8 heads for qPCR detection.

## **June 22****th****,2024**

1.  Feed preparation

Prepare 25 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for 250 larvaes of Spodoptera litura (10 larvae of Spodoptera litura in each box).

![](https://static.igem.wiki/teams/5044/notebook-6-22.png)

## **June 23****th****,2024**

1.  Feed preparation

Prepare 25 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for 250 larvaes of Spodoptera litura (10 larvae of Spodoptera litura in each box).

## **June 24****th****,2024**

1.  Feed preparation

Prepare 25 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for 250 larvaes of Spodoptera litura (10 larvae of Spodoptera litura in each box).

## **June 25****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replace the feed box of the hatchling larvae from the original 10-head box to 5-head box.

![](https://static.igem.wiki/teams/5044/notebook-6-25.png)

## **June 26****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvae of Spodoptera litura in each box).

## **June 27****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

3.  Observe, record the number of surviving Spodoptera litura, remove 5 Spodoptera litura from surviving Spodoptera litura for weight weighing, remove 8 heads for qPCR detection.

![notebook 6.27.png](https://static.igem.wiki/teams/5044/notebook-6-27.png)

## **June 28****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

## **June 29****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

3.  Observe, record the number of surviving Spodoptera litura, remove 5 Spodoptera litura from surviving Spodoptera litura for weight weighing, remove 8 heads for qPCR detection.

![notebook 6.29(2).png](https://static.igem.wiki/teams/5044/notebook-6-29-2.png)![notebook 6.29.png](https://static.igem.wiki/teams/5044/notebook-6-29.png)

N=26；N=26；N=26；N=27；N=26                 Sample：8

![notebook 6.29(1).png](https://static.igem.wiki/teams/5044/notebook-6-29-1.png)

## **June 30****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

## **July 1****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

![](https://static.igem.wiki/teams/5044/notebook-7-1.png)

## **July 2****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

![](https://static.igem.wiki/teams/5044/notebook-7-2.png)

## **July 3****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

![](https://static.igem.wiki/teams/5044/notebook-7-3.png)

## **July 4****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

## **July 5****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

![](https://static.igem.wiki/teams/5044/notebook-7-5.png)

## **July 6****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura (5 larvaes of Spodoptera litura in each box).

![notebook 7.6.png](https://static.igem.wiki/teams/5044/notebook-7-6.png)

## **July 7****th****,2024**

1.  Feed preparation

Prepare 50 small plastic boxes with a pinhole on the lid of the plastic box for breathability and artificial feed (KEYUN BIOCONTROL) the Spodoptera litura Placed in a 50mL syringe, squeeze into each small plastic box. Each time the feed is squeezed about 1cm long and pressed to pave, Using pipettes to absorb 200μL 5 groups of fungus on feed, and make a mark, open the lid naturally dry 30min ~ 1h. After the artificial feed fully absorbs the bacterial fluid can be tested.

2.  Biological Detection

Replacement of feed box for larvaes of Spodoptera litura 5 larvaes of Spodoptera litura in each box.

3.  Stability test of intestinal juice of *Spodoptera litura* to amiRNA.

* RNA from the B2 strains expressing the pMJ5 (ami*CHS1)*and pMJ7 (MS2-ami*CHS1)* plasmids was extracted.Make it into crude pure liquid （amiRNA/amiRNA+MS2）.
* The RNase of 5th instar larvae was extracted and diluted (1μl RNase+19ul water).
* RNase and crude pure fluid (amiRNA/amiRNA+MS2) were nursed at a ratio of 1: 1.
* The content of amiRNA was detected by Northern blot.

![notebook 7.7.png](https://static.igem.wiki/teams/5044/notebook-7-7.png)

Figure 2.Northern blot The expression of miRNA in the bacteria was examined.(Sample loading, 5 μ g.)

Experimental result:

The results showed that amiCHS1 was obviously degraded after 5 min incubation and completely degraded for a long time after 10 min. AmiCHS1 coated with MS2 coat protein can still be detected after 10min incubation (Figure 2). The results show that the CP of MS2 phage can protect amiRNA well. Subsequently, we will test the control effect of amiRNA on Spodoptera litura.

## **July 8****th****,2024**

Prepare 25 sandy plastic boxes with a small amount of feed (no bacterial solution) and a certain amount of moisture, prick holes in the lid of the plastic box with pinholes to make them breathable, and put 5-6 instar Spodoptera litura, which has been fed with different bacterial solutions, and wait for them to pupate.

![](https://static.igem.wiki/teams/5044/notebook-7-8.png)

## **July 9****th****,2024**

Prepare 25 sandy plastic boxes with a small amount of feed (no bacterial solution) and a certain amount of moisture, prick holes in the lid of the plastic box with pinholes to make them breathable, and put 5-6 instar Spodoptera litura, which has been fed with different bacterial solutions, and wait for them to pupate.

## **July 10****th****,2024**

Prepare 25 sandy plastic boxes with a small amount of feed (no bacterial solution) and a certain amount of moisture, prick holes in the lid of the plastic box with pinholes to make them breathable, and put 5-6 instar Spodoptera litura, which has been fed with different bacterial solutions, and wait for them to pupate.

## **July 11****th****,2024**

Prepare 25 sandy plastic boxes with a small amount of feed (no bacterial solution) and a certain amount of moisture, prick holes in the lid of the plastic box with pinholes to make them breathable, and put 5-6 instar Spodoptera litura, which has been fed with different bacterial solutions, and wait for them to pupate.

## **July 12****th****,2024**

Prepare 25 sandy plastic boxes with a small amount of feed (no bacterial solution) and a certain amount of moisture, prick holes in the lid of the plastic box with pinholes to make them breathable, and put 5-6 instar Spodoptera litura, which has been fed with different bacterial solutions, and wait for them to pupate.

## **July 13****th****,2024**

Take out the Spodoptera litura, which has pupated in the sandy soil, and weigh and photograph it.

![notebook 7.13.png](https://static.igem.wiki/teams/5044/notebook-7-13.png)

## **July 14****th****,2024**

Observe and record the pupation status of Spodoptera litura, and wait for it to emerge from the pupa and become an adult.

## **July 15****th****,2024**

Observe and record the pupation status of Spodoptera litura, and wait for it to emerge from the pupa and become an adult.

## **July 16****th****,2024**

Observe and record the pupation status of Spodoptera litura, and wait for it to emerge from the pupa and become an adult.

## **July 17****th****,2024**

Observe and record the pupation status of Spodoptera litura, and wait for it to emerge from the pupa and become an adult.

## **July 18****th****,2024**

The pupation of Spodoptera litura is basically completed. Observe and record the number of deformed adults and take photos for record.

## **July 19****th****,2024**

The pupation of Spodoptera litura is basically completed. Observe and record the number of deformed adults and take photos for record.

![](https://static.igem.wiki/teams/5044/notebook-7-19.png)

## **July 20****th****,2024**

The pupation of Spodoptera litura is basically completed. Observe and record the number of deformed adults and take photos for record.

All the data were summarized to calculate the pupation rate, deformity rate and mortality rate of Spodoptera litura fed with different bacterial fluids, and plotted into a pie plot as follows.

![notebook 7.20.png](https://static.igem.wiki/teams/5044/notebook-7-20.png)

## **July 22****th** **,2024:** The Expression Vector pMJ11 Was Designed Based on pYY25

**1.   The pFJ11 is made by the senior sister and we replaced the target gene on this basis**

![](https://static.igem.wiki/teams/5044/notebook-7-22.png)![](https://static.igem.wiki/teams/5044/notebook-7-22-1.png)

## **July 23****th** **,2024:** Two Primers were Designed and Synthesized by Wuhan Gencreate Company in China

XbaI-amiCHS1-F

AAAATCTAGANACATGAGGATCACCCATGTGAGGAATTCACCTCGTC

XhoI-amiCHS1-R

AAAACTCGAGTTCTAAGGGGTGCTGCCTGCGATCGATG

## **July 24****th** **,2024:** Get the Interest Fragment

Received two primers (Primer1: XbaI-amiCHS1-F Primer2: XhoI-amiCHS1-R) synthesized by the company.

**2.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| XbaI-amiCHS1-F | 2 μl |
| XhoI-amiCHS1-R | 2 μl |
| Golden Star T6 Super PCR Mix (Beijing Tsingke Biotechnology Co., Ltd., China) | 46 μl |
| Total volume | 50 μl |

**3.  Prepare with 1 % agarose gelatin.**

**4.  Electrophoresis.**

**5.  Take a picture of the gel.**

![](https://static.igem.wiki/teams/5044/notebook-7-24.png)

**6.  Recycling of DNA from Agarose Gels.**

Get the interest fragment amiCHS1 containing digestion sites (XhoI XbaI).

## **July 25****th** **,2024:** Plasmid Digestion

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| XbaI | 1 μl |
| XhoI | 1 μl |
| pYY25 | 15 μl |
| 10X QuickCut Green Buffer | 5 μl |
| ddH2O | 28 μl |
| Total volume | 50 μl |

**6.  37 ℃, 5h.**

**7.  Agarose gel preparation for electrophoresis.**

**8.  Electrophoresis.**

**9.  Recycling of DNA from agarose gels.**

![notebook 7.25.png](https://static.igem.wiki/teams/5044/notebook-7-25.png)

**10.  Measure nucleic acid concentration.**

**11.  Store at -20 °C for later use.**

## **July 26****th** **,2024:** the Plasmid and Fragment are Ligated by the T4 DNA Ligase

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| processed vector pYY25 | 1 μl |
| processed interest fragment amiCHS1 | 2.5 μl |
| T4 DNA Ligase | 0.5 μl |
| 10 × T4 DNA ligase Buffer | 1 μl |
| ddH2O | 5 μl |
| Total volume | 10 μl |

**2.  16 ℃, 3 h.**

**3.  The constructed plasmid was transformed into DH5α by heat shock.**

**·**Add 5 μl of the constructed plasmid mixture and place on ice for 20 min.

·Heat shock 42 ℃, 45 s.

·Add 500 μl of LB medium to each of the tubes.

·Shake at 211 rpm, 37 ℃ for 1 h.

**4.  Add 300 μl of the above transformation mix to a solid LB containing Kana (100 μg /ml) in a clean bench.**

**5.  Overnight at 37 °C.**

## **July 27****th** **,2024:** Check whether the Vector is Successfully Constructed

**1.  Colony PCR:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| 10 µM Primer F | 2 µl |
| 10 µM Primer R | 2 µl |
| Golden Mix (green) | 45 µl |
| Bacterial liquid | 1 µl |
| Total volume | 50 µl |

**2.  Agarose gel preparation for electrophoresis.**

**3.  Electrophoresis.**

![](https://static.igem.wiki/teams/5044/notebook-7-27.png)

## **July 28****th** **,2024:** Single Colony Expansion Culture

**1.  Pick the sequenced single colonies in the clean bench and add them to a bacterial bottle (contains 5 ml of liquid medium containing KanR).**

**2.  Overnight on a shaker at 37 °C.**

## **July 29****th** **,2024:** Plasmid Extraction

**1.  The bacterial fluid in the bacterial bottle appears cloudy.**

**2.  Plasmid Extraction (Operate according to the instructions).**

**3.  Measure nucleic acid concentration.**

**4.  Take 10 μl for sequencing.**

**5.  The company emailed back that the fragment sequenced yesterday was correct. Then the vector pMJ11 was constructed successfully.**

![notebook 7.30.png](https://static.igem.wiki/teams/5044/notebook-7-30.png)

## **July 31****th** **,2024:** The Expression Vector pMJ12 Was Designed Based on pYY25

**1.  The pFJ12 is made by the senior sister and we replaced the target gene on this basis**

![](https://static.igem.wiki/teams/5044/notebook-7-31.png)![](https://static.igem.wiki/teams/5044/notebook-7-31-1.png)

## **August 1****th** **,2024: Four Primers were Designed and Synthesized by Wuhan Gencreate Company in China**

NcoI-Cry1C-F

AAAACCATGGATGGAGGAGAACAATCAGAACCAGTG

ApaI-Cry1C-R

GGGCCCCTACTTTTGTGCTCTTTCAAGGTC

AfeI-amiCHS1-F

AAAAAGCGCTTGGATACTCTAGAACATGAGGATCACCCATGTNGAGGAATTCACCTCGTC

XhoI-amiCHS1-R

AAAACTCGAGNTTCTAAGGGGTGCTGCCTGCGATCGATGATTAATG

FrameSeq4-F

CGGAAGGCCGAGAGCTAACTATC

ZTYW2-R

TACGGGACGTCAGTGGCAGTTG

## **August 2****th** **,2024:** Get the Interest Fragment

Received four primers (Primer 1: NcoI-Cry1C-F Primer 2: ApaI-Cry1C-R Primer 3: AfeI-amiCHS1-F Primer 4: XhoI-amiCHS1-R) synthesized by the company.

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| NcoI-Cry1C-F | 2 μl |
| ApaI-Cry1C-R | 2 μl |
| Golden Star T6 Super PCR Mix (Beijing Tsingke Biotechnology Co., Ltd., China) | 46 μl |
| Total volume | 50 μl |

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| AfeI-amiCHS1-F | 2 μl |
| XhoI-amiCHS1-R | 2 μl |
| Golden Star T6 Super PCR Mix (Beijing Tsingke Biotechnology Co., Ltd., China) | 46 μl |
| Total volume | 50 μl |

**2.  Prepare with 1 % agarose gelatin.**

**3.  Electrophoresis.**

**4.  Take a picture of the gel.**

![notebook 8.1.png](https://static.igem.wiki/teams/5044/notebook-8-1.png)

**5.  Recycling of DNA from Agarose Gels.**

Get two kinds of interest fragment (Cry1C amiCHS1)

## **August 3****th** **,2024:** Plasmid Digestion

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| NcoI | 1 μl |
| ApaI | 1 μl |
| pYY25 | 15 μl |
| 10X QuickCut Green Buffer | 5 μl |
| ddH2O | 28 μl |
| Total volume | 50 μl |

**2.  37 ℃, 5h.**

**3.  Agarose gel preparation for electrophoresis.**

**4.  Electrophoresis.**

**5.  Recycling of DNA from agarose gels.**

![notebook 8.3.png](https://static.igem.wiki/teams/5044/notebook-8-3.png)

**6.  Measure nucleic acid concentration.**

**7.  Store at -20 °C for later use.**

## **August 4****th** **,2024:** the Plasmid and Fragment are Ligated by the T4 DNA Ligase

**1.  Reaction system:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| processed vector pYY25 | 1 μl |
| processed interest fragment Cry1C | 2.5 μl |
| T4 DNA Ligase | 0.5 μl |
| 10 × T4 DNA ligase Buffer | 1 μl |
| ddH2O | 5 μl |
| Total volume | 11 μl |

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| processed vector pYY25 | 1 μl |
| processed interest fragment amiCHS1 | 2.5 μl |
| T4 DNA Ligase | 0.5 μl |
| 10 × T4 DNA ligase Buffer | 1 μl |
| ddH2O | 5 μl |
| Total volume | 10 μl |

**2.  16 ℃, 3 h.**

**3.  The constructed plasmid was transformed into DH5α by heat shock.**

**·**Add 5 μl of the constructed plasmid mixture and place on ice for 20 min.

·Heat shock 42 ℃, 45 s.

·Add 500 μl of LB medium to each of the tubes.

·Shake at 211 rpm, 37 ℃ for 1 h.

**4.  Add 300 μl of the above transformation mix to a solid LB containing Kana (100 μg /ml) in a clean bench.**

**5.  Overnight at 37 °C.**

## **August 5****th** **,2024:** Check whether the Vector is Successfully Constructed

**1.  Colony PCR:**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| 10 µM FrameSeq4-F | 2 µl |
| 10 µM ZTYW2-R | 2 µl |
| Golden Mix (green) | 45 µl |
| Bacterial liquid | 1 µl |
| Total volume | 50 µl |

**2.  Agarose gel preparation for electrophoresis.**

**3.  Electrophoresis.**

![notebook 8.5.png](https://static.igem.wiki/teams/5044/notebook-8-5.png)

## **August 6****th** **,2024:** Single Colony Expansion Culture

**1.  Pick the sequenced single colonies in the clean bench and add them to a bacterial bottle (contains 5 ml of liquid medium containing KanR).**

**2.  Overnight on a shaker at 37 °C.**

## **August 7****th** **,2024:** Plasmid Extraction

**1.  The bacterial fluid in the bacterial bottle appears cloudy.**

**2.  Plasmid Extraction (Operate according to the instructions).**

**3.  Measure nucleic acid concentration.**

**4.  Take 10 μl for sequencing.**

**5.  The company emailed back that the fragment sequenced yesterday was correct. Then the vector pMJ12 was constructed successfully.**

![](https://static.igem.wiki/teams/5044/notebook-8-7.png)

![notebook 7.7(1).png](https://static.igem.wiki/teams/5044/notebook-7-7-1.png)

## **August 9****th** **,2024:** Particle bombardment

1.  Gold particles are co-precipitated with DNA containing the target gene, usually with the selection marker gene.

2.  Gene guns (such as PDS1000/He particle delivery systems) are used to accelerate and bombard DNA-coated gold particles into plant tissues. Precise control of bombardment parameters is required to ensure that DNA can effectively enter plant cells without causing excessive damage.

3.  Cut the bombarded leaves into small pieces and place them on spectinomycin-containing medium to screen for cells containing transgenes.

![](https://static.igem.wiki/teams/5044/notebook-8-9.png)

## **August 10****th** **,2024:** Screened resistant shoots

1.  Place the bombarded leaf segments on the regeneration medium to select for transformed cells containing the resistance gene.

2.  After a period of cultivation, the resistant tissue will gradually regenerate to form new plantlets. During this process, the tissue undergoes dedifferentiation and redifferentiation, eventually leading to the formation of complete plants.

3.  Perform PCR analysis on the regenerated plants to detect whether the target gene has been integrated into the plant genome.

![notebook 8.10.png](https://static.igem.wiki/teams/5044/notebook-8-10.png)

## **August 17****th** **,2024:** southern blot

![notebook 8.11.png](https://static.igem.wiki/teams/5044/notebook-8-11.png)

## **August 25****th** **,2024:** Screening of homoplasmy

1.  Further confirm the homozygous state of transgenic plants through seed screening and plant regeneration. This typically involves sowing the seeds of transgenic plants on a medium containing a selection agent to test whether all offspring plants exhibit resistance.

2.  Perform genetic analysis on the regenerated transgenic plants and their offspring to ensure that the chloroplast genome of all plants is homogenized, meaning that each chloroplast contains the same foreign gene.

3.  Conduct phenotypic observations on the transgenic plants and their offspring to ensure that their growth and development are similar to those of wild-type plants, without any negative phenotypic effects.

April 22th, 2024: Selection of Target Genes

*Spodoptera litura is an important crop pest, which has evolved a
high degree of resistance to all insecticides targeted at it.
After 24 h injection of 500 ng/μL dsRNA, the expression levels of
SlCHS1 and SlCHS2 were significantly inhibited in S. litura (Yu et
al.,2020). Among them, SlCHS1 has a high expression level in the
outer epidermis and is exclusively expressed in the epidermis
underlying the cuticular exoskeleton and related ectodermal cells
such as tracheal cells, while SlCHS2 is highly expressed in the
midgut, which protects the intestinal epithelium from mechanical
disruption and invasion by various pathogens.*

*These results suggest that SlCHS1 might play an important role
during S. litura larval molting, while SlCHS2 has no significant
effect on S. litura molting. Therefore, we have chosen SlCHS1 for
further study.*

DNAMAN

DNAMAN is a one-for-all software package for molecular biology applications. This package provides an integrated system with versatile functions for high efficiency sequence analysis. It can also perform multiple sequence alignment, designing PCR primers, protein sequence analysis or drawing plasmids.![](https://www.lynnon.com/images/desktopwin.png)

siDirect

siDirect (http://design.RNAi.jp/) is a web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi). Highly effective siRNA sequences are selected using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity. Our efficient software avoids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook. The website accepts an arbitrary sequence as input and quickly returns siRNA candidates, providing a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing.![](https://tse4-mm.cn.bing.net/th/id/OIP-C.81N3fH9VuHiw_QA0DnNIqQHaDG?rs=1&pid=ImgDetMain)

Wizard

Chitin Synthase (CHS1 and CHS2)

**Chitin synthases** are enzymes that catalyze the synthesis of chitin, a key component of the exoskeletons of arthropods, including insects. In the context of **Spodoptera litura**, **SlCHS1** and **SlCHS2** refer to two specific chitin synthase genes. **SlCHS1** is highly expressed in the outer epidermis and related ectodermal cells, while **SlCHS2** is primarily expressed in the midgut, playing roles in protection against mechanical disruption and pathogen invasion.

Double-Stranded RNA (dsRNA)

**Double-stranded RNA (dsRNA)** is a form of RNA that consists of two complementary strands. In the context of gene silencing, dsRNA can be used to trigger the RNA interference (RNAi) pathway, leading to the degradation of specific messenger RNAs (mRNAs) and the subsequent inhibition of gene expression.

Artificial microRNA (amiRNA)

**Artificial microRNAs (amiRNAs)** are synthetic small RNA molecules designed to mimic natural microRNAs. They are engineered to target and silence specific genes through the endogenous miRNA pathway. The backbone of an amiRNA refers to the sequence structure that is used to carry the guide sequence for targeting the mRNA of interest.

Backbone Selection

**Backbone selection** in the context of amiRNA design involves choosing the most effective pre-microRNA (pre-miR) structure from different organisms. The choice of backbone can influence the efficiency of gene silencing, with some backbones being more effective than others.

Bioinformatics Tools

**Bioinformatics tools** are computational programs and databases used to analyze biological data, such as DNA, RNA, and protein sequences. These tools can help predict the most effective fragments for designing amiRNAs, ensuring high specificity and minimal off-target effects.

Polymerase Chain Reaction (PCR)

**Polymerase chain reaction (PCR)** is a technique used to amplify specific DNA segments. It involves repeated cycles of heating and cooling to denature, anneal, and extend DNA, resulting in the exponential amplification of the target DNA sequence.

Agarose Gel Electrophoresis

**Agarose gel electrophoresis** is a method used to separate DNA, RNA, or proteins based on their size. The sample is loaded into wells in an agarose gel, and an electric current is applied, causing the molecules to migrate through the gel. Smaller molecules move faster and travel farther than larger ones.

DNA Extraction and Purification

**DNA extraction and purification** involve isolating DNA from a sample, such as a bacterial culture, and removing contaminants. This process typically includes cell lysis, followed by the addition of buffers to precipitate and purify the DNA.

T4 DNA Ligase

* T4 DNA ligase is an enzyme that catalyzes the formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate groups of two DNA fragments, joining them together. This enzyme is commonly used in molecular biology for the construction of recombinant DNA.

Colony PCR

**Colony PCR** is a method used to screen bacterial colonies for the presence of a specific DNA insert. A small portion of each colony is used as a template for PCR, and the resulting products are analyzed to determine if the desired DNA fragment has been successfully cloned.

Gene Silencing

**Gene silencing** is a process by which the expression of a gene is reduced or completely inhibited. This can be achieved through various mechanisms, such as RNA interference (RNAi), where **double-stranded RNA (dsRNA)** triggers the degradation of specific messenger RNAs (mRNAs).

Insecticides

* **Insecticides** are substances used to kill or control insects. They can be natural or synthetic and are commonly used in agriculture to protect crops from pests. Insects, like **Spodoptera litura**, may develop **resistance** to insecticides, necessitating the development of alternative control methods.

Resistance

**Resistance** in the context of pest management refers to the ability of an organism to withstand the effects of a substance that is normally toxic to it. For example, **Spodoptera litura** has developed a high degree of resistance to many insecticides, making traditional control methods less effective.

Epidermis

The **epidermis** is the outermost layer of cells covering an organism, including the skin of animals and the cuticle of plants. In insects, the epidermis is part of the exoskeleton and plays a crucial role in protection and support.

Midgut

The **midgut** is a part of the digestive tract in many animals, including insects. It is involved in the digestion and absorption of nutrients. In **Spodoptera litura**, the midgut is protected by chitin synthase 2 (SlCHS2), which helps maintain the integrity of the intestinal epithelium.

Mechanical Disruption

**Mechanical disruption** refers to physical damage to tissues or organs, which can occur due to external forces or internal processes. In the context of the midgut, mechanical disruption could result from the passage of food or the presence of pathogens.

Pathogens

**Pathogens** are organisms that cause disease. In the context of **Spodoptera litura**, pathogens could include bacteria, viruses, or fungi that invade the midgut and potentially cause harm to the insect.

Molting

* **Molting** is the process by which an insect sheds its old exoskeleton to allow for growth and the formation of a new one. This process is critical for the development of larvae into adults and is influenced by the activity of genes like **SlCHS1**.

Electrophoresis

**Electrophoresis** is a laboratory technique used to separate molecules based on their size and charge. In the context of molecular biology, agarose gel electrophoresis is commonly used to separate DNA fragments.

Gelatin Agarose

**Agarose** is a polysaccharide extracted from seaweed and is used to make gels for electrophoresis. **Gelatin agarose** typically refers to the preparation of an agarose gel, which is used to visualize and analyze DNA fragments after electrophoresis.

DNA Quantification

* **DNA quantification** is the process of measuring the concentration of DNA in a sample. This is important for ensuring that there is enough DNA for downstream applications, such as PCR, cloning, or sequencing.

Transformation Mix

A **transformation mix** is a solution containing plasmid DNA and other reagents that is used to introduce the DNA into bacterial cells. The mix is often prepared with a buffer and sometimes includes heat shock to facilitate the uptake of the plasmid by the bacteria.

Heat Shock

**Heat shock** is a method used in bacterial transformation where the cells are briefly exposed to high temperatures (typically 42°C) to increase the permeability of the cell membrane, allowing the uptake of plasmid DNA.

Luria-Bertani (LB) Medium

**Luria-Bertani (LB) medium** is a nutrient-rich broth or agar used for the growth of bacteria. It contains tryptone, yeast extract, and sodium chloride, and is often used in microbiology and molecular biology for culturing *E. coli* and other bacteria.

Kanamycin

**Kanamycin** is an antibiotic used to select for bacteria that have been successfully transformed with a plasmid carrying a kanamycin resistance gene. Only those bacteria that have taken up the plasmid will survive in the presence of kanamycin.

Recombinant DNA

**Recombinant DNA** is DNA that has been created by combining DNA sequences from different sources, often using restriction enzymes and DNA ligases. Recombinant DNA technology is used to create new genetic combinations that do not exist naturally.

Expression Vector

An **expression vector** is a type of plasmid or virus that is designed to carry a gene of interest into a host cell and promote the expression of that gene. Expression vectors typically contain regulatory elements, such as promoters and enhancers, to control the level and timing of gene expression.

Syringe

A **syringe** is a medical tool used to inject or withdraw fluids from the body. In the context of the experiment, a 50mL syringe is used to dispense artificial feed into small plastic boxes.

Pipette

A **pipette** is a laboratory tool used to measure and transfer precise volumes of liquid. In the experiment, pipettes are used to apply 200μL of fungal solution onto the feed.

Fungus (Fungi)

**Fungi** are a kingdom of organisms that include yeasts, molds, and mushrooms. They play a key role in the decomposition of organic matter and can also be used as biological control agents. In this experiment, five groups of fungi are applied to the feed to test their effects on **Spodoptera litura** larvae.

Biological Detection

* Biological detection involves the use of living organisms or their components to detect or measure the presence or effect of another substance. In the experiment, it refers to the process of observing and measuring the impact of the fungi on the larvae.

qPCR (Quantitative Polymerase Chain Reaction)

**qPCR** is a laboratory technique used to amplify and simultaneously quantify a targeted DNA molecule. It allows for the measurement of gene expression levels and is used here to detect and quantify specific genetic material in the **Spodoptera litura** larvae, possibly to assess the effects of the fungal treatment.

Larvae

**Larvae** are the juvenile form of many animals, especially insects, that undergo complete metamorphosis. In this experiment, the larvae of **Spodoptera litura** are the subjects being treated and observed.

Bacterial Fluid

**Bacterial fluid** may refer to a suspension containing bacteria or bacterial products. In the context of the experiment, it seems to be a term used to describe the fungal solution, although typically, bacteria and fungi are distinct types of microorganisms.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

Contents

## **Engineering Success**

Thoughts makes difference – this is what the Monkey King always said when he encountered difficulties on the Journey to the West, and it is also the most important point in the engineering process. Read more to find out about our engineering cycles, and see how we grew from mini monkeys to another Great Sage Equaling to Heaven.

## **Insect Feeding and Bioassays**

### **Cycle 1**

* **Design**

After we have built the engineered strain, we continued to feed insect and bioassays as planned. We consulted information on the culture of *Spodoptera litura*, and carried out biological tests in a dedicated insect room.

* **Construction**

Prepare a small plastic box, use a pinhole to pierce the lid of the plastic box to make it breathable, put the artificial feed of *S. litura* (made by Keyun Company) in a 50mL syringe (note to remove the needle), squeeze artificial feed into each small plastic box, squeeze the feed about 1cm long each time and press it flat, use a pipette to absorb 200μL of bacterial liquid on the feed, open the lid and air dry it naturally for 30min~1h, and wait for the artificial feed to completely absorb the bacterial solution before the biological test can be carried out. The hatched larvae were placed in a small feed box (10 *S. litura* larvae were placed in each box), and the data were weighed on various days of *S. litura*, and samples were taken to record its growth.

* **Test**

When experimenting, we operate strictly according to the design conditions. We monitor the preservation conditions of the larvae to ensure the stability of environmental factors. The early stages of the experiment went well, but during the larval pupation stage, we encountered an unexpected challenge: an unusually high larval mortality rate, a phenomenon that was also common in the control group. Despite our best efforts to simulate ideal pupation conditions, larval mortality is still high. This led us to rule out the potential effects of Artificial microRNAs (amiRNAs), as the larvae were unable to normalize pupation even without introducing any exogenous agents.

* **Learn**

We recognize that successful engineering requires a deep understanding of the mechanisms by which things break down or fail. During the experiment, we found that *S. litura* did not feed when pupating. After reviewing the literature, we found that we ignored the key factor that *S. litura* needs to pupate in sandy soils. Under natural conditions, *S. litura* nests and pupates in sandy soils, which our experimental environment does not provide. In this case, we do not fully understand the specific environmental needs of *S. litura*. This information suggests that we may need to rethink our feeding strategy.

### **Cycle 2**

* **Design**

In this cycle of experiments, we will replace the feed with sand in the rearing container to simulate the natural pupation environment of *S. litura*. We will also adjust the feeding density to reduce interfering with the larvae and optimize the feed formula to provide a more complete range of nutrition. With these improvements, we hope to be able to effectively reduce the mortality of larvae and successfully promote their pupation and emergence.

* **Construction**

Increase the amount of feed appropriately in the early stage of insect rearing. The larvae of *S. litura* pupate around 25 days. The amount of larval feed is significantly reduced. At this point, sand will be used instead of feed in a rearing container to simulate the natural pupation environment of *S. litura* so that it can pupate smoothly.

* **Test**

During the experiment, the pupation of *S. litura* in the control group was normal, and the results of the amiRNA group showed pupation deformity, as shown in Figure 1. The effectiveness of the experiment was successfully verified.

![](https://static.igem.wiki/teams/5044/20241001015836.png)

**Figure 1** *S. litura* feed on the following strains of bacteria: CK (empty vector pET28a control), VLP (VLP-expressing bacteria), amiRCHS1 (amiRCHS1-expressing bacteria), VLP-amiRCHS1 (VLP and amiRCHS1 co-expressing bacteria). Scale bar: 1 cm.

* **Learn**

Through an iterative cycle of "design-build-test-learn", we learn from failure and improve the design of experiments. After learning the key information that *S. litura* needs sand to pupate, we adapted the protocol and successfully pupated the larvae and emerged into adults. This process not only enhances our understanding of the *S. litura*'s life cycle, but also demonstrates the importance of learning from failure and continuous improvement in bioengineering.

## **Plastid transformation**

### **Cycle 1**

* **Design**

Agrobacterium transformation is a method widely used in plant transgenics. It relies on the T-DNA region of Agrobacterium and can insert foreign genes into the chromosomal DNA of plant cells. However, this approach is primarily aimed at the transformation of the nuclear genome rather than the chloroplast genome.

Gene gunning is an effective method for chloroplast transformation. This method allows foreign DNA to pass through the chloroplast's double membrane and integrate into the chloroplas t genome. That's why we chose to use a gene gun for quality transliteration.

However, as undergraduates, we still wanted to verify whether Agrobacterium could perform plastid transformation in tobacco, so we carried out an experimental design.

* **Construction**

We transformed the constructed plant expression vector into an Agrobacterium strain. Plant cells were infected with Agrobacterium, transformed plant cells were cultured, and positive plants were screened.

* **Test**

During the testing phase, we found that tobacco leaves transformed with Agrobacterium could not be grown on antibiotic-containing media, suggesting that Agrobacterium transformation is very inefficient or not feasible in terms of plastid transformation. This result is consistent with our expectation that Agrobacterium is not suitable for plastid transformation.

![微信图片_20240930194724.jpg](https://static.igem.wiki/teams/5044/20240930194724.jpg)

**Figure 2** PCR was performed on the experimental leaves, but the results showed that the target gene was not detected.

* **Learn**

Faced with the failure of the experiment, we conducted a thorough analysis. We revisited the data and confirmed that Agrobacterium transformation was indeed not suitable for plastid transformation, and that chloroplast transformation needed to take into account the high copy number of the chloroplast genome and the challenge of having foreign DNA pass through the inner and outer membrane barriers of chloroplasts. Gene gun conversion is a better option.

### **Cycle 2**

* **Design**

After confirming that Agrobacterium transformation was not suitable for plastid transformation, we decided to use gene gun technology for plastid transformation. Cut the transformed leaves into small pieces and screen on spectinomycin-containing RMOP medium to obtain a self-contained transfection system.

* **Construction**

Tobacco plastid transformation was performed. Briefly, young leaves of aseptically grown tobacco plants were bombarded with DNA-coated 0.6 μm gold particles using a PDS-1000/He Biolistic Particle Delivery System (Bio-Rad). Bombarded leaves were cut into ~5 × 5-mm pieces and selected on RMOP medium containing 500 mg L−1 spectinomycin. Several independent transplastomic lines were obtained and subjected to two to three additional rounds of regeneration on spectinomycin-containing medium to select for homoplasmy.

![微信图片_20240925211822.jpg](https://static.igem.wiki/teams/5044/20240925211822.jpg)

**Figure 3** Schematic diagram of gene gun bombardment.

* **Test**

Due to our lack of experience, we first tested the bombardment with pressure, and finally the 1100psi pressure bombardment was the best, so we chose 1100psi for the gene gun transformation. During the transformation process, different bombardment distances of 6 cm, 9 cm, and 12 cm were used for incubation and PCR, and the results are shown in Figure 4. Tobacco leaves with a bombardment distance of 9 cm grew better on bispecific antibody medium.

![image.png](https://static.igem.wiki/teams/5044/image.png)![微信图片_202410010341134.png](https://static.igem.wiki/teams/5044/202410010341134.png)

**Figure 4** (A)Tobacco leaves with bombardment distances of 9 cm, 6 cm, 12 cm, and 12 cm grown on bispecific antibody medium. (B) Only the 1st and 5th showed the target band, i.e. the blade at a bombardment distance of 9 cm, was successfully transformed.

* **Learn**

In the course of our test, we found that the bombardment distance of the gene gun has a very important effect on the chloroplast transformation effect, and the mass conversion test at different bombardment distances under the same pressure is significantly different.

From this cycle, we have a better understanding of the chloroplast transformation of the plasmid, which will guide us to the next cycle.

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5044/header-result.jpg)

Contents

## **Engineered bacteria construction**

### **Prediction of Effective Fragments of CHS1**

The deduced amino acid sequences of *SlCHS1* and *SlCHS2* were analyzed by using DASTAR software. The open reading frame (ORF) was predicted according to the ORF finder tool (<https://www.ncbi.nlm.nih.gov/orffinfer/>). The molecular weight (MW) and isogenic point (pI) of *SlCHS1* and *SlCHS2* were calculated using ExPASy (http://web.expasy.org/compute\_pi). The signal peptides of *SlCHS1* and *SlCHS2* were predicted using SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP). The functional domain was predicted by using SMART software (http://smart.embl-heidelberg.de/). The membrane-spanning domain was predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The phylogenetic tree was constructed with MEGA 7.0 software using the neighbor-joining method with 1000-fold bootstrap resampling. Protein sequences from different insect species were obtained from GenBank (http://www.ncbi.nlm.nih.gov/) and used in the phylogenetic analysis.

![](https://static.igem.wiki/teams/5044/result-16.jpg)**Figure 1**Multiple sequence alignment of the conserved catalytic domain of the chitin synthases (CHSs) from three insect species. CHSs are from *Spodoptera litura* (*Sl*), *Spodoptera exigua* (*Se*) and *Helicoverpa armigera* (*Ha*). Seven characteristic motifs (M 1–7) for insect chitin synthases are indicated with red boxes.

### **Construction of bacteria expression vectors**

The bacteria expression vectors were constructed based on pET28a. Three vectors were designed for expressing amiR-CHS1, VLP ( HIV TAT-conjugated coat protein (CP) dimer), and VLP plus amiR-CHS1 ( VLP-amiR-CHS1, Figure 3) in *Escherichia coli*.

![微信图片_20240930012352.png](https://static.igem.wiki/teams/5044/20240930012352.png)**Figure 2**Vector design for expressingamiRCHS1, VLP and VLP-amiRCHS1 in bacteria. pac: the site where CP recognizes RNA and assembles it; TAT, transmembrane peptide. The above vectors are all modified from pET28a.

### **Detection of RNA Expression**

Three bacteria expression vectors were expressed in *E.coli* BL21(DE3) that lacks RNase III activity (BL21Δrnc). RNA in different strains before and after IPTG induction (overnight) was extracted and detected with formaldehyde denaturing gel. Northern blot showed the expression amiR-CHS1 in BL21Δrnc that expresses amiR-CHS1 and VLP-amiR-CHS1.

![微信图片_20240929235243.png](https://static.igem.wiki/teams/5044/20240929235243.png)

**Figure 3**Detection of amiRNA accumulation in BL21ΔRNC. After IPTG induction, amiRNA expression was detected in the strains expressing VLP, amiR-CHS1 and VLP-amiR-CHS1 (138 nt).  Samples of 10 µg total cellular RNA were loaded in each lane of the RNA blot. The Gelview-stained gel before blotting is shown below the blot as a loading control.

### **Detection of Protein Expression**

Proteins were extracted from different BL21Δrnc strains before and after IPTG induction (overnight). Successful expression of VLP was determined by SDS-PAGE in bacteria expressing VLP and VLP-amiR-CHS1.

In summery, both protein and amiRNA could efficiently express in BL21Δrnc.

![微信图片_20240930000118.png](https://static.igem.wiki/teams/5044/20240930000118.png)

**Figure 4**  Detection of protein expression level in BL21ΔRNC. After IPTG induction, the expression of 2×CP (28 kDa) was detected in the VLP and VLP-amiR-CHS1-expressing strains. Sample load, 10 μg.

## **Insect feeding and bioassays**

### **VLP improved RNAi efficiency in** ***S. litura***

Bioassay was conducted via feeding *S. litura* larvae with an artiﬁcial diet supplemented with the engineered bacteria. Only the VLP-amiR-CHS1 markerdly reduced the larval weight gain7 days after feeding (DAF).

![](https://static.igem.wiki/teams/5044/20241001172757.png)

**Figure 5**  Bioassay of *S. litura* larvae 7 days after feeding (DAF).  (A) Representative examples of *S. litura* larvae. (B) Larval weight of *S. litura*. Data are means ± SE (n ≥ 30).

### **Relative expression of** ***SlCHS1***

As expected, the expression of target gene was significantly reduced in larvae fed with VLP-amiR-CHS1-expressed bacteria compared with other groups.

![](https://static.igem.wiki/teams/5044/20241001173945.png)

**Figure 6** Analysis of *CHS1* expression in *S. litura* after 7 d of feeding by qRT-PCR. Data are means ± SEM (n ≥6). Different lowercase letters above the columns indicate significant differences (*P* < 0.05, one-way ANOVA with Tukey’s multiple range test).

### **Detection the stability of the amiRNA**

To test whether the MS2 VLP could reduce the degradation of amiRNA by nuclerase in the midgut of *S. litura*, RNA from different BL21Δrnc strains was extracted and digested with intestinal fluid. The results showed that naked amiR-CHS1 was quickly degraded after 5 min of incubation and completely degraded after 10 min. By contrast, the amiCHS1 encapsulated by the MS2 VLP could be detected even after 10 minutes of incubation. These results indicate that the MS2 VLP could protect amiRNA from degradation in the intestinal fluid of *S. litura.*

![](https://static.igem.wiki/teams/5044/result-9.jpg)

**Figure 7**Stability test of *S. litura*'s intestinal fluid against amiRNAs. Northern blotting of amiR-CHS1 in engineered *E. coli* extract after incubation with intestinal fluid (15-fold dilution) of tobacco cutworms at room temperature. Naked amiR-CHS1 is strongly degraded after 5 minutes and completely degraded after 10 minutes. In contrast, amR-CHS1 filled with VLP is relatively stable. Sample load, 5 μg.

### **Silencing** ***CHS1*** **caused developmental defect of** ***S. litura***

Bioassay of *S. litura* was performed till the molting stage.  The pupation and molting of *S. litura* larvae were significantly inhibited by feeding the VLP-amiR-CHS1.

![](https://static.igem.wiki/teams/5044/20241001125444.png)

**Figure 8** Effects on pupation and molting of S. litura after silencing SlCHS1. (A,B) Phenotype of S. litura 25 and 32 DAF.

Compared with CK ,VLP and amiR-CHS1, VLP-amiR-CHS1 was more effective in affecting the pupation rate and deformity rate of *S. litura*.

![](https://static.igem.wiki/teams/5044/result-12-1.png)

**Figure 9**Representation (%) of developmental and mortality rates of S. litura larvae until molting stage after feeding on engineered bacteria.

## **Production of transplastomic tobacco plants**

### **Plastid transformation vector construction**

These results indicated that the expression of amiRNA in bacteria could effectively interfere with the growth and development of *S. litura*. Subsequently, **PLASTID**  PESTICIDES*™* constructed plastid transformation vectors pMJ11 and pMJ12 expressing amiRNAs.

![微信图片_20240930000333.png](https://static.igem.wiki/teams/5044/20240930000333.png)

**Figure 10**Tobacco plastid transformation vector. The selectable marker gene *aadA* is driven by the *psbA* promoter (*Cr*PpsbA) and the 3'UTR of the rbcL gene (CrTrbcL) of *Chlamydomonas reinhardtii*. G*accD*: tobacco accD promoter; Prrn: tobacco rRNA operon promoter; TrrnB: *rrnB* terminator of *E. coli*.

### **Biolistic transformation**

Sterile tobacco leaves were bombarded with plastid plasmids using a 1100 psi rupture disk at target distance of 9 cm. Following the biolistic bombardment, the leaf samples were diced into 5 × 5 mm and were placed on regeneration media including 500 mg/L spectinomycin.

Authentic transplastomic clones carrying an *aadA* gene are resistant to both spectinomycin and streptomycin, whereas spontaneous spectinomycin-resistant mutants are resistant only to spectinomycin. While resistance is manifested as formation of green calli with regenerating shoots, sensitivity is indicated by formation of scanty white callus in the leaves. Resistance to streptomycin and spectinomycin indicates the presence of selectable aadA gene. However, double selection delays shoot formation. Hence, we scored the presence of *aadA* gene by resistance to streptomycin plus spectinomycin, but screen homoplastic transplastomic plants on spectinomycin.

**Table 1**. Summary of plastid transformation experiments conducted in tobacco.

![](https://static.igem.wiki/teams/5044/1352.png)

![](https://static.igem.wiki/teams/5044/20241001123329.png)

**Figure 11**Screening of leaves transformed with dual antibiotics. (A) Three groups of tobacco leaves transformed with the vector Nt-amiRCHS1 (B) Four groups of tobacco leaves transformed with the vector Nt-VLP-amiRCHS1.

### **Homoplasy confirmation of transplastomic plants by Southern blot**

![](https://static.igem.wiki/teams/5044/1210.png)

![](https://static.igem.wiki/teams/5044/1211.png)

**Figure 12**Generation of transplastic tobacco plants expressing MS2 VLPs for delivery of amiRCHS1 against *S. litura*. (A) Physical map of the targeted region in the tobacco plastid genome (ptDNA) and maps of the transformed region of the tobacco plastid genome in the Nt-VLP, Nt-amiRCHS1, and Nt-VLP-amiRCHS1 transplastic lines. The SphI restriction sites used for restriction fragment length polymorphism (RFLP) analysis of transplastomic lines are indicated, and the sizes of the restriction fragments detected in Southern blot analyses are given. (B) Southern blot analysis of plastid transformation of tobacco line. SphI-digested DNA samples were separated by electrophoresis in a 1% agarose gel and hybridized with the DIG-labeled probes shown in (A). The absence of the 4.9 kb hybridization signal of the wild-type tobacco plastid genome indicated that all transplastic lines reached homoplasmy.

![](https://static.igem.wiki/teams/5044/1234.png)

**Figure 13**Stable inheritance of plastid transgenes and wild-type-like phenotypes of transplastomic tobacco plants. (A) Seed assays to confirm homoplasmy of transplastomic tobacco plants. Seeds obtained from wild-type (Nt-wt) and transplastomic plants were germinated on synthetic medium containing spectinomycin (500 mg L−1). Resistance of seedlings to the antibiotic and lack of segregation confirm the homoplasmic state of the transplastomic lines. Phenotypes of transplastomic tobacco lines grown in soil for 3 (B) and 6 (C) weeks. Scale bars: 1 cm in (A) and 10 cm in (B,C).

## **Reference**

[1] Li S, Kim DS, Zhang J. Plastid-mediated RNA interference: A potential strategy for efficient pest control. Plant Cell Environ. 2023 46:2595-2605.

[2] Zhang, S., An, S., Li, Z., Wu, F., Yang, Q., Liu, Y. et al. (2015) Identification and validation of reference genes for normalization of gene expression analysis using qRT‐PCR in *Helicoverpa armigera* (Lepidoptera: Noctuidae). Gene, 555, 393–402.

[3] Wu M, Dong Y, Zhang Q, et al. (2022) Efficient control of western flower thrips by plastid‐mediated RNA interference. Proceedings of the National Academy of Sciences, 119, e2120081119.

[4] Rana S., Rajurkar, A.B., Kumar, K.K. & Mohankumar, S. (2020) Comparative analysis of chitin synthaseA dsRNA mediated RNA interference for management of crop pests of different families of lepidoptera. Frontiers in Plant Science, 11, 427.

[5] Kim, D.S. & Zhang, J. (2023) Strategies to improve the efficiency of RNAi‐ mediated crop protection for pest control. Entomologia Generalis, 43, 5–19.

[6] Singh, I.K., Singh, S., Mogilicherla, K., Shukla, J.N. & Palli, S.R. (2017) Comparative analysis of double‐stranded RNA degradation and processing in insects. Scientific Reports, 7, 17059.

[7] Bao, W., Cao, B., Zhang, Y. and Wuriyanghan, H. (2016) Silencing of Mythimna separata chitinase genes via oral delivery of in planta-expressed RNAi effectors from a recombinant plant virus. Biotechnol. Lett. 38, 1961–1966.

[8] Diao, Pengfei, Zhang, Qimeng, Sun, Hongyu, Ma, Wenjie, Cao, Aiping, Yu, Ruonan, Wang, Jiaojiao et al. (2019) miR403a and SA are involved in NbAGO2 mediated antiviral defenses against TMV infection in Nicotiana benthamiana. Genes 10, 526–542.

[9] Huan, L., Tianming, L., Dongming, F., Furong, G., Hong, L.W., Wei, G., Xiao, F.C. et al. (2019) Chromosome level draft genomes of the fall armyworm, *Spodoptera frugiperda* (Lepidoptera: Noctuidae), an alien invasive pest in China. BioRxiv. 671560–671580.

[10] Liu, J., Smagghe, G. and Swevers, L. (2013) Transcriptional response of BmToll9-1 and RNAi machinery genes to exogenous dsRNA in the midgut of Bombyx mori. J. Insect Physiol. 59, 646–654.

[11] Chen, S., Hofius, D., Sonnewald, U. & Börnke, F. (2003) Temporal and spatial control of gene silencing in transgenic plants by inducible expression of double‐stranded RNA. Plant Journal, 36, 731–740.

[12] Kumar, M., Gupta, G.P. & Rajam, M.V. (2009) Silencing of acetylcholinesterase gene of *Helicoverpa armigera* by siRNA affects larval growth and its life cycle. Journal of Insect Physiology, 55, 273–278.

[13] Zhang, Q., Dong, Y. & Zhang, J. (2023) Host‐induced silencing of MpPar6 confers Myzus persicae resistance in transgenic rape plants. Journal of Integrative Agriculture.

[14] Chen, Q., Shen, P., Bock, R., Li, S. & Zhang, J. (2022) Comprehensive analysis of plastid geneexpression during fruit development and ripening of kiwifruit. Plant Cell Reports, 41, 1103–1114.

[15] Baum, J.A., Bogaert, T., Clinton, W., Heck, G.R., Feldmann, P., Ilagan, O., Johnson, S. et al. (2007) Control of coleopteran insect pests through RNA interference. Nat. Biotechnol. 25, 1322–1326.

[16] Eamens, A.L., Smith, N.A., Curtin, S.J., Wang, M.B. and Waterhouse, P.M. (2009) The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA, 15, 2219–2235.

[17] Schwab, R., Ossowski, S., Riester, M., Warthmann, N. and Weigel, D. (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell, 18, 1121–1133.

[18] Terenius, O., Papanicolaou, A., Garbutt, J.S., Eleftherianos, I., Huvenne, H., Kanginakudru, S., Albrechtsen, M. et al. (2011) RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. J. Insect. Physiol. 57, 231–245.

Prediction of Effective Fragments of CHS1

* **Sequence Analysis**: The amino acid sequences of *SlCHS1* and *SlCHS2* (chitin synthase 1 and 2 from *Spodoptera litura*) were analyzed using DASTAR software. The open reading frames (ORFs) were predicted using the ORF Finder tool, while molecular weight (MW) and isoelectric point (pI) were calculated with ExPASy. Signal peptides were predicted by SignalP 4.1, and functional domains were identified using SMART. Membrane-spanning regions were predicted with TMHMM Server v. 2.0.
* **Phylogenetic Analysis**: A phylogenetic tree was constructed using MEGA 7.0, based on the neighbor-joining method with 1000 bootstrap resamplings, including sequences from different insect species obtained from GenBank.

Plasmid Construction

* **Vectors Design**: E. coli expression vectors were designed based on pET28a. These included:
* **Transformation**: The constructed vectors were transferred into *E. coli* B2 (BL21ΔRNC).

Detection of RNA Expression

* **Northern Blotting**: RNA expression was detected using formaldehyde denaturing gel and Northern blot. The results showed that amiRNA expression was present in strains expressing VLP, amiR-CHS1, and VLP-amiR-CHS1 after IPTG induction.

Detection of Protein Expression

* **SDS-PAGE**: Protein expression was assessed using SDS-PAGE. The expression of 2×CP (28 kDa) was confirmed in strains expressing VLP and VLP-amiR-CHS1, indicating efficient expression of both protein and amiRNA.

Insect Feeding and Bioassays

* **Growth Assay**: Hatched larvae of *S. litura* were fed with BL21ΔRNC strains containing different vectors. Larvae were weighed at specific time points, and growth status was recorded. Mortality rates were also monitored.
* **Relative Expression of SlCHS1**: qPCR was used to detect the relative expression of the target gene *SlCHS1* after feeding with different BL21ΔRNC strains.

Stability of amiRNA

* **Degradation Test**: To assess the stability of amiRNA, RNA from *E. coli* B2 expressing pMJ5 plasmid was incubated with *S. litura* intestinal fluid. Naked amiRNA was significantly degraded after 5 minutes, whereas amiRNA encapsulated by MS2 CP remained stable even after 10 minutes, indicating protection against RNase degradation.

Pupal Weight and Development

* **Pupation Assay**: Sixth-instar larvae of *S. litura* were placed on sandy soil, and pupal development was monitored. Pupa weights were measured, and deformity and mortality rates were counted. Malformation rates were also observed during the moth stage.

Production of Transplastomic Tobacco Plants

* **Vector Construction**: Based on the successful interference with *S. litura* growth, PLASTID PESTICIDES™ constructed plastid transformation vectors pMJ11 and pMJ12 to express amiRNAs in tobacco plants.
* **Biolistic Transformation**: Tobacco leaves were transformed using biolistic methods, and transformants were selected on a medium containing double antibiotics.
* **Homoplasy Confirmation**: Southern blot analysis was used to confirm the homoplasmy of transplastomic plants. Resistant buds were then transferred to rooting medium to induce rooting, resulting in dsRNA-producing transplastomic tobacco plants.

Figures

* **Figure 1**: Multiple sequence alignment of the conserved catalytic domain of chitin synthases from three insect species, highlighting characteristic motifs.
* **Figure 2**: Schematic representation of the constructed vectors: amiR-CHS1, VLP, and VLP-amiR-CHS1.
* **Figure 3**: Northern blot showing amiRNA accumulation in *E. coli* B2 strains.
* **Figure 4**: SDS-PAGE gel demonstrating the expression of 2×CP in *E. coli* B2 strains.
* **Figure 5**: Growth curve of *S. litura* after 7 days of feeding with engineered *E. coli*.
* **Figure 6**: Relative expression of *SlCHS1* in *S. litura* after 7 days of feeding.
* **Figure 7**: Northern blot showing the stability of amiRNA in *S. litura* intestinal fluid.
* **Figure 8**: Pupal deformity of *S. litura* on the 25th day.
* **Figure 9**: Feathering malformation in *S. litura* on the 32nd day.
* **Figure 10**: Schematic of the tobacco plastid transformation vector expressing amiRNA.
* **Figure 11**: Screening of biolistically transformed tobacco leaves on double antibiotics.
* **Figure 12**: Southern blot confirming the homoplasmy of transplastomic tobacco plants.
* **Figure 13**: Rooted transplastomic tobacco plants producing dsRNA.

{% endblock %}