Team:FAFU-CHINA/Proof
Bioassay
1.Introduction
In our project, we transform the expression vector of Cry and Cyt toxins. To compare the toxicities, we also tested the toxicity of Bacillus thuringiensis strain LLP229. The larvae Aedes albopictus is our experimental object.
2.Material, Condition and Method
The larvae of Ae. albopictus are feeding in the Key Laboratory of Biopesticide and Chemical Biology. The generation of Ae. albopictus is 11. The LLP229 strain of Bacillus thuringiensis is provided by Key Laboratory of Biopesticide and Chemical Biology. The concentration of LLP229 is recorded in each table.
The CC530 and FA479 strains of Chlamydomonas reinhardtii and TAP medium are bought from the Aquatic Species Research Institute in Wuhan, Hubei. The concentration of Chlamydomonas reinhardtii is recorded in each table.
We contrasted various toxins group expression vectors and transform them into Chlamydomonas reinhardtii as following groups: Cry4Aa+2A+Cyt1, Cry4Aa+2A+Cyt2, Cry10Aa+2A+Cyt1, Cry10Aa+2A+Cyt2, Cry11Aa+2A+Cyt1, Cry11Aa+2A+Cyt2.
The toxicity analysis condition is in an environmentally controlled room (T=28±1 °C, RH=85%±5%, L: D=14:10).
Bioassay of various concentrations were assayed against 30 third-instar larvae in 20 ml of dechlorinated water. Each group was replicated 3 times. The level of mortality was recorded after 48 hours. Then calculated the corrected mortality. The bioassay was then repeated such that a concentration range yielding 10–90% mortality was obtained and the 50% lethal concentration (LC50) was calculated.
Table 1.1 Toxicity of LLP29 to A. albopictus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 | 30 | 30 | 100 |
| 2 | 30 | 30 | 100 |
| 3 | 30 | 30 | 100 |
| Control | 30 | 0 | 0 |
Note. Then concentration of LLP29 is 2.16 x 109 (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =2.7A.
Table 1.2 Toxicity of LLP29 to Culex quinquefasciatus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 | 30 | 30 | 100 |
| 2 | 30 | 30 | 100 |
| 3 | 30 | 30 | 100 |
| Control | 30 | 0 | 0 |
Note. Then concentration of LLP29 is 2.16 x 109 (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =2.7A)
Table 1.3 Toxicity of LLP29 to Culex quinquefasciatus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 | 30 | 30 | 100 |
| 2 | 30 | 30 | 100 |
| 3 | 30 | 30 | 100 |
| Control | 30 | 0 | 0 |
Note. Then concentration of LLP29 is 2.16 x 109 (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =2.7A)
Table 2.1 Toxicity of Chlamydomonas reinhardtii (Cry4Aa+2A+Cyt1) to A. albopictus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 |
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| 2 |
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| 3 |
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| Control 1 Control 2 |
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Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =xxxx)
2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)
3. The Control 2 group is TAP medium (Concentration =?)
Table 2.2 Toxicity of Chlamydomonas reinhardtii (Cry4Aa+2A+Cyt2) to A. albopictus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 |
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|
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| 2 |
|
|
|
| 3 |
|
|
|
| Control 1 Control 2 |
|
|
|
Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =xxxx)
2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)
3. The Control 2 group is TAP medium (Concentration =?)
Table 2.3 Toxicity of Chlamydomonas reinhardtii (Cry10Aa+2A+Cyt1) to A. albopictus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 |
|
|
|
| 2 |
|
|
|
| 3 |
|
|
|
| Control 1 Control 2 |
|
|
|
Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =xxxx)
2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)
3. The Control 2 group is TAP medium (Concentration =?)
Table 2.4 Toxicity of Chlamydomonas reinhardtii (Cry10Aa+2A+Cyt2) to A. albopictus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 |
|
|
|
| 2 |
|
|
|
| 3 |
|
|
|
| Control 1 Control 2 |
|
|
|
Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =xxxx)
2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)
3. The Control 2 group is TAP medium (Concentration =?)
Table 2.5 Toxicity of Chlamydomonas reinhardtii (Cry11Aa+2A+Cyt1) to A. albopictus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 |
|
|
|
| 2 |
|
|
|
| 3 |
|
|
|
| Control 1 Control 2 |
|
|
|
Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =xxxx)
2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)
3. The Control 2 group is TAP medium (Concentration =?)
Table 2.5 Toxicity of Chlamydomonas reinhardtii (Cry11Aa+2A+Cyt2) to A. albopictus
| Repeating group | Number of insects | Number of mortality | Corrected mortality (%) |
| 1 |
|
|
|
| 2 |
|
|
|
| 3 |
|
|
|
| Control 1 Control 2 |
|
|
|
Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD600 of 1.0 = 8 x 108 cells/ml, OD600 =xxxx)
2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)
3. The Control 2 group is TAP medium (Concentration =?)
Reference
1.Zhang, L., Huang, E., Lin, J., Gelbič, I., Zhang, Q., Guan, Y., … Guan, X. (2010). A novel mosquitocidal strain LLP29 isolated from the phylloplane of. Microbiological Research, 165(2), 133–141. doi: 10.1016/j.micres.2009.03.002
