Part:BBa_K4627002
Ceres
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2012
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 994
Illegal AgeI site found at 1978
Illegal AgeI site found at 2041 - 1000COMPATIBLE WITH RFC[1000]
Origin(organism)
Galleria mellonella
Ceres trial expression
This 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.
NCBI:XP_026756459.1
- 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 Ceres
- 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.(Figure 2)
Figure 2. Group of Ceres trial expression
- 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 3, the C3 control group produced the most protein, whose incubated temperature is 37 ℃, and the induced temperature is 16℃ with 3mM IPTG.
Figure 3. From the SDS-page, it can be seen that the C3 experimental group secretes the most protein
So we finally decided to use the concentration and induction temperature set by C3 to culture the 900ml large system of later mutations in order to obtain more protein.
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