Since secretory express of cry protein in yeast is crucial for the elimination of A. cantonensis, we fuses a signal peptide with cry protein. In order to find the signal peptide of higher secretion efficiency, we employ RFP as the reporting molecule to reflect the effectiveness of 4 different signal peptides, namely JF, JFm, SCMa and OPT. According to the experiment results, JFm is the optimal option amongst. Afterwards, cry protein secretory expression in S. cerevisiae is accomplished through combined use with JFm.
Regarding the distinguished secretory capability of JFm, the implementation of signal peptide JFm can also be used for reference of the part by future teams that has the demand for secretory protein expression. (Fig.1)
Fig.1 Verification of SP-RFP/Cry (A) SDS-page analysis of SP-Cry expression. 1:JF-His-Cry, 2:JFm-His-Cry; 3:ScMα-His-Cry; 4:OPT-His-Cry; 5:JF-Cry-His; 6:JFm-Cry-His; 7:ScMα-Cry-His; 8:OPT-Cry-His. (B) SDS-page analysis of SP-RFP expression. 1:JF-RFP; 2:JFm-RFP; 3:ScMα-RFP; 4:OPT-RFP. (C) Fluorescence intensity of RFP in supernates from samples with different signal peptides. (D) The fluorescence indication of different signal peptides.
M:marker; C:control, CENPK2-1C; S:supernate; P:precipitate
Cry1518-35, a type of Insecticidal Crystal Proteins formed during the development of Bacillus thuringiensis(Bt), is chosen for the specified elimination of A. cantonensis. We have successfully expressed Cry1518-35 in both E.coli (Proven by protein gel & Western blot) and Saccharomyces cerevisiae (Proven by protein gel), while the A. cantonensis elimination trial is under active progression. our successful attempt to kill nematodes with cry protein provides future teams with the idea of using other Bt produced ICPs to apply elimination of high specificity. (Fig.2)
Fig.2 Cry1518-35 protein expression in E.Coli ; (A) Cry protein from B.thuringiensis. (B) Construction of Cry protein with 6x His tag. (C) SDS-page analysis of Cry protein with His tag. (D) Western Blot analysis of Cry protein with His tag. M:marker; 1:PET28a-His-Cry; 2:PET28a-Cry-His; C:control, 1&2 without induction; W:whole cell; S:supernate; P:precipitate. (E) Toxicity assay of Cry protein with C. elegans.
In the enzymatic pathway that produces isopentanol, ARO10 and ADH7, which encode ketoacid decarboxylase and alcohol dehydrogenase respectively, have been demonstrated to significantly boost the conversion of KIC to isopentanol when overexpressed. Moreover, overexpressing ATF1 has been found to augment the production of isoamyl acetate from isopentanol. In our project, we innovatively attempted to co-overexpress ARO10, ADH7, and ATF1, successfully amplifying isoamyl acetate production. This not only confirmed the feasibility and efficacy of this strategy but also paved a new, efficient avenue for future projects that necessitate the production of isoamyl acetate. (Fig.3) (Fig.4)
Fig.3 Expression of isoamyl acetate in S. cerevisiae. (A) Genetic pathway of producing isoamyl acetate. ARO10, encoding 2-oxoacid decarboxylase. ADH7, encoding for alcohol dehydrogenase 7. ATF1, encoding for alcohol acetyl-coenzyme A (acetyl-CoA) transferase (AATase) (B) Genetic circuit construction for producing isoamyl acetate. ARO10, ADH7, and ATF1 are transformed into site 106 through homologous recombination using 106 upstream (106 US) and 106 downstream (106 DS). (C) Gel electrophoresis analysis of integrated sequence ARO10-ADH7-ATF1
Fig.4 GC-MS analysis of the product isoamyl acetate of SCIE L1, using Standard isoamyl acetaet as control
We have implemented a highly stable fermentation system for a more predictable isoamyl acetate yield.
1.The transformants were streaked on YPD+2% glucose plates and incubated at 30˚C.
2.Then the tubes containing 5mL YPD medium supplemented with 2% glucose were inoculated with fresh cell cultures of a single colony and incubated overnight at 30 °C, 200rpm in a shaker.
3.200ul of the pre culture are grown in 20mL YPD with 2% glucose and 2% dodecane for 48 hours at 30 °C and 200 rpm.
4.After fermentation, dodecane layer was obtained through centrifugation. Anhydrous sodium sulfate was then added to the collected dodecane and the mixture was held for 2 hours.
5.The supernatant layer of dodecane is collected for gas chromatography-mass spectrometry (GC–MS) analysis.
In the GC-MS analysis, the split ratio was set at 5:1, injection port temperature 150°C. GC oven temperature was initially held at 40 °C for 1 min, ramped with a gradient of 6 °C/min until 140 °C. The temperature was then raised with a gradient of 20 °C/min to 180 °C and held for 4 min.
The results of the modified strains SCIE L1, SCIE L2 fermentation was analyzed using GC-MS (Fig.5)
Fig.5 Testing the production of isoamyl acetate (A) standard curve of isoamyl acetate (B) the production of isoamyl acetate of SCIE L1 and SCIE L2.
When expressing cry protein in Escherichia coli, we tried alternate expression vectors, including PET28a, PW1, pSEVA321 and pSEVA331.
Fig.6 The genetic circuits for Cry+6x His tag in different vectors
Induced expression of the protein and SDS-page analysis were carried out respectively after the construction of vectors, with different protein expression efficiency correspondingly. We discovered that PW1 and pSEVA331 are more efficient than other vectors.
This investigation has reference value in E.coli vector selection for heterologous gene expression for iGEM community.
Fig.7 SDS-page analysis for expression in different vectors. (A) 1: 321-His-Cry; 2: 331-Cry-His; 3: 331-His-Cry. (B) 1: PW1-Cry-His; 2: PW1-His-Cry; 3: 321-Cry-His.
(C) 1: PET28a-Cry-His in BL21; 2: PET28a-Cry-His in ROSETTA.
M: Marker; C: E.coli without induction; W: whole cell; S: supernate; P: precipitate;
The field of bioethics has consistently garnered significant attention and generated extensive public discourse. However, there persists a notable prevalence of misconceptions regarding bioethics among the public. To enhance our comprehension of bioethics, we engaged in a thorough discourse with Professor Guo, who was previously the cheif of BGI's Genomics' institutional review board, which resulted in a novel perspective on the subject matter. Under the guidance of Professor. Guo, we have compiled a handbook, which we hope will help the public to understand more about bioethics.
(Click Here for Downloading the PDF Document)
The objective of the development of this handbook is two-fold: firstly, to furnish individuals with a dependable and comprehensive resource that facilitates the expansion of their understanding in the field of bioethics; and secondly, to foster a well-informed and constructive public dialogue concerning these significant ethical matters. We possess a strong belief that this handbook will function as a highly beneficial resource for individuals such as students, healthcare professionals, researchers, policymakers, and anybody with an inclination towards exploring the ethical dimensions of biology study.
