Part:BBa_K4690002
EstA
Pyrethroids are set of bionic synthetic insecticides, with high efficiency and broad spectrum characteristics. Previous data showed that the insecticidal virulence of pyrethroids is 10 to 100 times higher than the old generation of insecticides such as organochlorine, organophosphorus, etc. Based on this, pyrethroids have been widely used in recent years. However, pyrethroids are highly toxic to fish, harmful to beneficial insects such as bees, and long-term repeated use may even lead to insect resistance. The iBowu team is concerned about the environmental problems caused by pyrethroids accumulation in water, and plans to use synthetic biological methods to degrade pyrethroids.
Part Collection
We have uploaded four basic parts for producing pyrethroids degradation enzymes, which have not been uploaded by previous iGEM teams, we also make an improvement of a previous part BBa_K4210004 with new data and new figures.
| Part number | Description | Chasis | Results | Species |
|---|---|---|---|---|
| BBa_K4690000 | EstPs1 | E. coli BL21 (DE3) | Success | Pseudomonas synxantha PS1 |
| BBa_K4690001 | EstP | E. coli BL21 (DE3) | Success | Klebsiella sp. strain ZD112 |
| BBa_K4690002 | EstA | E. coli BL21 (DE3) | Success | Bacillus cereus BCC01 |
| BBa_K4690003 | PytH | E. coli BL21 (DE3) | Success | Sphingobiumsp. strain JZ-1 |
Characterization by iBowu-China 2023
EstA-associated biodegradation of pyrethroids was confirmed in 2019, which used the key degrading gene estA and related carboxylesterase EstA from Bacillus cereus BCC01.
We used the following usual procedure to do the experiments:
1. Verification of the sequence. This part sequence we submitted was come from Bacillus cereus BCC01. We downloaded the sequence from NCBI and in order to be suitable for bacterial expression, we asked a biology company to undergo codon optimization and synthesize the target sequence.
2. We then constructed it into a pET28(+) plasmid and transformed the plasmids into E. coli BL21(DE3) strains.
3. After cultured on LB solid medium (Kanamycin) for 12 h, we picked a single colony into 4 ml LB medium (Kanamycin) and the mix was then shaken at 37℃ until OD600 = 0.6.
4. In order to set proper control, we divided the bacteria solution into three parts, for the first part, we added 0.5 mM IPTG and cultured in 16℃ for 20 h; the second part was added with 0.5 mM IPTG as well but cultured in 37℃ for 4 h. The last one added nothing to serve as a control.
5. When the expression process was finished, we centrifuged the bacterial solution at 12000 rpm, and the precipitation was resuspended with RIPA buffer to lysate bacteria, we also added loading buffer to heat at 96℃ for 10 min, followed by SDS-PAGE and Coomassie brilliant blue staining for expression test.
According to our SDS-PAGE result, protein EstA showed clear bands around expected molecular weight. In order to confirm the band we pointed out indicated our target proteins, we did an interview with our teacher, who suggested western blot assays to provide evidence, so we asked for collaboration and the following are results we got, which provided us positive results.
In the figure, the left panels are SDS-Page results obtained by us. The right panels are Western Blot results obtained by our external help (Corresponding SDS-Page runs weren’t shown).
16C-1 means the bacteria was cultured at 16 degree Celsius and the supernatant was used for the test. 16C-2 means the 16 degree Celsius culture and the precipitation was used. 37C-1 means the bacteria was cultured at 37 degree Celsius and the supernatant was used for the test. 37C-2 means the 37 degree Celsius culture and the precipitation was used.
We then wondered whether we could promote the expression of EstA through condition adjustment, so we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. We found 20℃ lead to the highest EstA expression level.
The next experiment we did was to fixed the induction temperature and IPTG concentration, then set a series of time gradient. This time we showed EstA expressed pretty well under 20℃ and the induction time seems not influence the expression level.
Compare Enzymic Activity
After successfully expressing five types of pyrethroid degradation enzymes, we induced the expression of these proteins again under the optimal conditions obtained in the previous experiments. We then mixed the 2 ml bacterial solution with 0.5 ml of specified concentration pyrethroid solution and subjected it to 4 hours of rotational mixing. To conveniently and quickly detect the pyrethroid content within the system, we used commercially available test strips. As shown in the figure below, compared to the control group, all five experimental groups exhibited the ability to degrade pyrethroids. Among them, the degradation effect of enzyme EstA was the most significant, followed by enzyme PytH, and then Pyre1, while the degradation effects of enzyme EstPS1 and EstP were similarly weak, i.e., poor degradation effect. One red line on the top indicates positive test outcome, meaning the test sample contains pyrethroid. Two red lines indicate negative test outcome, meaning no (or less) pyrethroid in the test sample.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |