Part I: Test pAC-LYC plasmid
The pAC-LYC plasmid is available from Addgene, and contains three genes from Erwinia herbicola: CrtE, CrtI, and CrtB. This plasmid is provided by Addgene on an agar stab transformed into Top10 E. coli. That means that it can simply be streaked onto LB Agar with chloramphenicol to make lycopene-producing colonies that should be pink to red in color.
| Contain CrtE,CrtI,CrtBAddgene |
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To be updated
Part II: Design strategies for increasing lycopene production
Using tools such as Biocyc and KEGG, select enzymes that can be added to (or knocked out of) your lycopene-producing E. coli to increase the amount of lycopene they produce. The pAtipiTrc plasmid available in Addgene represents one possible strategy, as it overexpresses a heterologous idi enzyme. Labs working with PCR and cloning may want to try replacing the idi gene in pAtipiTrc with alternative enzymes to see how they impact colony color.
Theory Task
Select enzymes that can be added to your lycopene-producing E. coli to increase the amount of lycopene they produce.
- Lycopene biosynthesis pathway was constructed by integration of CrtE,ErtB, and CrtI.
- According to study of Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering, Blakeslea trispora (BtCrtl) had excellent performance on lycopene production and lycopene proportion on carotenoid.Blaskeslea trispora is a fungal plant pathogen of genus in Blakeslea.
| Approach of this reading |
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'''
Through screening CrtE, CrtB and CrtI from diverse species, an optimal carotenogenic enzyme combination was obtained, and CrtI from Blakeslea trispora (BtCrtI) was found to have excellent performance on lycopene production as well as lycopene pro- portion in carotenoid. '''
To be updated
Lab Task
Labs working with PCR and cloning may want to try replacing the idi gene in pAtipiTrc with alternative enzymes to see how they impact colony color.
| pAtipiTrc |
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To be updated
Part III: Design strategies for converting lycopene to beta-carotene
Theory Task
Use Biocyc and/or KEGG to identify the enzyme that can convert the red pigment lycopene to the orange pigment beta-carotene.
- I researched beta-carotene pathway firstly KEGG.
- Then clicked "metabolic pathway"
- Researching "beta-carotene" and "lycopene", "gamma-Carotene" are located between them. It means their pathway is "lycopene" --> "gamma-Carotene" --> "beta-carotene".
- These pathway both mentions "CrtY". Therefore checked enzyme of both "gamma-Carotene" --> "beta-carotene".
Both has this enzyme "EC 5.5.1.19". She has several names as follows.
- lycopene beta-cyclase;
- CrtL;
- CrtL-b;
- CrtY;
- LCYb;
- carotenoid beta-end group lyase (decyclizing)
| Beta-carotene | Pathway-1 | Pathway-2(Red circles are "Beta-carotain") |
|---|---|---|
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| Pathway-3 | Gamma-Carotene | CrtY |
|---|---|---|
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Lab Task
Plasmids that can be co-transformed with pAC-LYC that produce this enzyme can be found in Addgene. Labs working on miniprep and transformation can order these plasmids to observe switch the color of their E. coli from red to orange.
- As result of reaserch "lycopene beta-cyclase" which is another name of Crt, there are two plasmid which contain lycopene beta-cyclase.
| pAtLCYbSK | pAC-BETA-At |
|---|---|
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To be updated
Weekly Reflection:
Do your activities this week raise new ethics and/or safety considerations you had not considered in week 1? Describe what activities have raised these considerations and any changes you have implemented in response.
- KEGG is very useful tool to get information about pathway and enyme. I was proud of such very nice database is being organized in Japanese University. Though several lecture of Bio Academy, there are variety of tools and database which can be used free of charge. Thefore I think that Bio's learnig cost is very low while wet material is high cost which means that easy to get information in order to use some bad purpose. If we make big accident without appropriate understanding, such chance might be dissapeared. Therefore, I think that it should be taken care to understand properly as one of bio hacker.