Part:BBa_K4627099

Demetra

Demetra is an enzyme that degrades polyethylene，belonging to the polyphenol oxidase group. It can realize the oxidation of C-C bond at room temperature, so as to promote its fracture. At present, although many microorganisms have been found to be able to use polyethylene as a carbon source, no other enzymes have been found that can degrade polyethylene. Demetra is reported to be more effiency compared with Ceres, but we have just do the test.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1018
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1018
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 278
  Illegal BglII site found at 1574
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1018
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1018
  
• 1000
  COMPATIBLE WITH RFC[1000]

Origin(organism)

Galleria mellonella

Structure Design

This sequence is optimized by Escherichia coli codon. Codon optimization is needed when expressed in different vectors.
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Demetra trial expression

We used wild protein for experimental expression, and by adding different concentrations of IPTG induction and different induction temperatures to the 50 ml system, we determined the most suitable IPTG concentration and induction temperature for protein secretion.
NCBI:XP_026756396.1

• 1. 1. We first constructed wild-type Ceres with XhoⅠ and NdeⅠ endonucleases. The plasmid is synthesized by the company and then returned.(Figure 1)
  

Figure 1. Atlas of Demetra

• 2. Design the IPTG concentration gradient
  

In order to find the most suitable temperature for protein secretion and IPTG content, we designed eight different experimental groups, and added an IPTG-free control group for the experiment.
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• 3. Expression in BL21 Escherichia coli
  

a. We express wild Ceres protein in BL21 E. coli and culture in a 50 mL system to obtain sufficient protein.
b. Use an ultrasonic sterilizer to break the bacteria and release the protein. As can be seen from the figure 2, there is no distinct banding. So, we take test continually.

Figure 2. From the SDS-page, it can be seen that there is no distinct banding.

• 4. As pET22b was not a suitable vector for Demetra, we selected different vectors and strains for experimental expression. We selected 7 vectors and 3 different strains for the test, and set 5 different IPTG concentrations(Figure 3). After inducing protein expression in the tube, SDS-page was used to verify the successful expression(Figure 4).
  

Figure 3. Map of Demetra constructed by different plasmids

Figure 2. Results of SDS-page

The results are shown in Table 1. pET15b and 28bs can’t be constructed, so we judged that they are not suitable as Demetra vectors. The vectors, strains and concentrations with good expression effect were pGEX-6P-1, BL21(DE3) and 0.7mM, respectively.

Table 1. Result of protein D test