Part:BBa_K4690000

EstPs1

This part encodes a novel carboxylesterase of Pseudomonas synxantha PS1 isolated from oil well-produced water, showed high biodegradability towards pyrethroid pesticides.

In iGEM 2023, iBowu_China had deep feelings about environmental pollution. We have saw many of the shocking numbers and pictures caused by environmental problem, which has been severely affecting our own lives. Although human beings invented advanced technology and great civilization, we have neglected to protect the natural environment. Pyrethroid accumulation, as a typical example, has caused significant threat to beneficial insects in aquatic ecosystems. So, how can we solve the problem?

This year, iBowu_China carefully searched papers and noticed some of the natural enzymes can be used in pyrethroid degradation, which further lead us to design the new project using synthetic biology approach to biodegrade pyrethroids.

Part Collection by iBowu-China 2023

This part is a member of the part collection to solve pyrethroid biological degradation. Please refer to the hub page for the collection BBa_K4690002.

Characterization by iBowu-China 2023

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]