Engineering

BBA_K4690000

A paper published on scientific reports mentioned the EstPS1 gene, which encodes a novel carboxylesterase of Pseudomonas synxantha PS1 isolated from oil well-produced water, showed high biodegradability towards pyrethroid pesticides.

We used the following usual procedure to do the experiments:

1. Verification of the sequence. This part sequence we submitted was come from Pseudomonas synxantha PS1. 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 EstPS1 can be expressed but precipitated in the inclusion body. 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 solubility of EstPS1 through condition adjustment, so we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. However, the SDS-PAGE result suggested that no matter what the temperature is, EstPS1 is left in the inclusion body.

The next experiment we did was to fixed the induction temperature and IPTG concentration, then set a series of time gradient. Luckily, this time the SDS-PAGE result suggested EstPS1 was expressed under 16℃, we observed little soluble bands after 3 h induction.

BBa_K4690001

Pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112, could not only degrade pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyze rho-nitrophenyl esters of various fatty acids, indicating EstP as an esterase with broad substrates.

We used the following usual procedure to do the experiments:

1. Verification of the sequence. This part sequence we submitted was come from Klebsiella sp. strain ZD112. 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 EstP can be expressed but precipitated in the inclusion body. 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.

We then wondered whether we could promote the solubility of EstP through condition adjustment, so we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. We found although EstP also precipitated in the inclusion body, but 22℃ expression expression produced more soluble protein.

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 more soluble EstP protein were produced after 24 h induction under the temperature of 22℃.

BBa_K4690002

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.

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.

he 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.

BBa_K4690003

PytH, isolated from Sphingobiumsp. strain JZ-1, encoding a pyrethroid-hydrolyzing carboxylesterase, which was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides.

We used the following usual procedure to do the experiments:

1. Verification of the sequence. This part sequence we submitted was come from Sphingobiumsp. strain JZ-1. 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 PytH 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.

We then wondered whether we could promote the expression of PytH through condition adjustment, so we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. We found 16℃ lead to the highest PytH expression level. As the temperature arises, PytH comes to the inclusion body, so it prefers lower temperature.

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 the highest PytH expression level was achieved with 18 h or 20 h induction under the temperature of 16℃.