Team:BHSF/Description

BHSF

We are here to win!

Passion

We are all looking forward to realise our program - manufacturing carminic acid using E.coli.
We are willing to put every effort into it.

Collaboration

We have a systemetic and exact plan of each one's jobs.
Everyone participates a lot.

Progression

We participate in IGEM for self-improvement and personal growth rather than only the prizes.

Project

Description, Design, Result, Implementation

Description

Carminic acid (CA) is widely used in dye production, the only way to extract CA is from cochineal insect. However, it takes about 90,000 insects to produce 1kg Carminic Acid. Using insects to produce CA leads to several animal issues, such as allergy, and religious prohibits.
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Design

The general idea behind this experiment is to use gene-edited E.coli to express gene fragments of polyketide synthase, cyclase, monooxygenase and C-glucosyltransferase. After a series of chemical reactions, gene edited E-coli can produce carminic red.

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Result and expectations

In our experiment, we know that we successfully expressed antBDEFG and DnrF. In the future, we can repeat this procedure in different kinds of E.coli that can tolerate the toxicity of IPTG, and shorten the time for induction. Since the usage of carminic acid is mostly abundant, we need to look into how to simplify and increasing the amount of production.

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Implementation

The reason for doing this project is that after knowing a paper and discussing it, the students in the same group are more interested in a real-life problem - the shortage of carmine acid.
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Design

1.The polyketide synthases used in the experiment include five enzymes, namely antd, ante, antf, antg and antb. With transgenic technology, the existing gene which can produce the enzymes illustrated above were transcribed into the plasmid of E. coli to express the required polyketide synthase. Polyketide synthase is used to catalyze the reaction of one acetyl coenzyme A and seven malonyl coenzymes A which are the common substances used in E. coli. The product of this reaction is octaketide intermediate, which can produce sek4 and sek4b. 2.In the cyclization process,two existing gene fragments are used to express Zhui and zhuj enzymes respectively. These two enzymes are cyclase. One of the branch products in the previous step, was catalysed by Zhui to form sek4, and the product was catalysed by zhuj to form the final product of cyclization.

3. There is one hydroxyl group and one glucose between flavokermesic acid and carminic acid. We transferred the gene fragment which can produce gtcgt and dnrf into E.coli plasmid. The gene fragment expressing gtcgt was extracted from Gentiana Triflora, and the gene fragment expressing dnrf was from Streptomyces peucetius. The product in the previous step was catalysed by these two enzymes in turn, and finally generated the required carminic acid.

4. In order to make the third step reaction more efficient, we mutated gtcgt and dnrf. The amino acid proline 217 of dnrf was mutated to lysine to form dnrfp217k; The 93rd valine of gtcgt was mutated to glutamine, and the 193rd tyrosine was mutated to phenylalanine, forming gtcgtv93q / y193f. So as to improve the reaction efficiency.

5. DNA electrophoresis, SDS-PAGE and liquid chromatography-mass spectrometry (lc-sm) will be used to detect the successful production of carminic acid.