While pinene is a relatively simple molecule requiring only the addition of one synthase gene, lots of high-value molecules have specific stereochemistry that is hard to synthesize in lab, but could be produced through this method. An example of something we’d like to work towards is the production of vincristine, a stereo-specific cancer drug used for childhood cancers.
We hope that overall, difficult to synthesize chemicals could be produced more efficiently through transgenic plants
Maybe we misspoke, the problem in isolating terpenes is more like environmental concerns rather than the compound itself being difficult to extract from the plant/tree. We’ll talk more about different medicinal and industrial uses in our live presentation!
We chose pinene in part because it requires only one synthase gene to be added, and also because (since terpenes are a major part of the cannabis industry), there are cheap ways to test for the presence and quantities of it.
Great Presentation! When you have the sticky ends, how come they are always AATT? Is that due to the restriction enzyme that you used or is there another reason for having the same sequence?
Hi Eitan, The AATT sticky ends are always true for EcoRI which is an example of a conventional restriction enzyme, where the cut site is in the middle of the recognition site. The enzymes we used, BsaI and AarI are type 2 restriction enzymes where the sticky ends can be any 4-piece combination of the 4 base pairs. This is because BsaI and AarI cut outside of the recognition site.
We will transfer our final vector plasmid from E. Coli to an agrobacterium – a type of bacteria that infects plants. From there we will allow it to infect the soybeans and incorporate the engineered plasmid into the soybean genome
Great Presentation!! When you mentioned the sticky ends you said that it was always AATT? Is that due to the restriction enzyme that you used and will it always be like that?
Thanks for asking this! We didn’t really have time to explain that in the video. Because the recognition site is removed when the new plasmid is created, there is no “scar” on the new plasmid. This means we can just slap on the recognition site anywhere onto any sequence we need, without having the recognition site in the end product. Whereas with the reversible conventional enzymes, you would need to conveniently have the recognition site for those already part of the sequence.
There’s varying levels of accuracy for each method and ease of execution:
Sequencing is the most accurate, but it takes the most time to send off and we have to pay so it would be inefficient to use it for every step.
The antibiotic resistance is good to make sure the growths have the plasmid but it’s not guaranteed that all the colonies have the pinene gene and the antibiotic resistance so it’s much lower accuracy, but a good way to track steps.
The gel electrophoresis is a good middle ground because it’s more accurate than the resistance, but it can be done in lab at the time.
The point of terpenes is to allow plants to interact with their outside world- it may attract pollinators, deter pests, help plants recover from injuries, or make them tasty or not tasty to predators/things that would spread their seeds. They can be citrusy, earthy, spicy, etc. If you think about the wide variety of smells found in essential oils- those are comprised of lots and lots of terpenes.
When you mention that this research could contribute to broader industrial applications, what specific examples do you see this being applied to?
While pinene is a relatively simple molecule requiring only the addition of one synthase gene, lots of high-value molecules have specific stereochemistry that is hard to synthesize in lab, but could be produced through this method. An example of something we’d like to work towards is the production of vincristine, a stereo-specific cancer drug used for childhood cancers.
We hope that overall, difficult to synthesize chemicals could be produced more efficiently through transgenic plants
You mentioned that terpenes have medicinal and industrial uses, but are very difficult to isolate… How often are they used in medicine/industry?
Maybe we misspoke, the problem in isolating terpenes is more like environmental concerns rather than the compound itself being difficult to extract from the plant/tree. We’ll talk more about different medicinal and industrial uses in our live presentation!
What an interesting topic! What made you choose this topic? I know you mentioned why you choose soybeans but why pinene?
We chose pinene in part because it requires only one synthase gene to be added, and also because (since terpenes are a major part of the cannabis industry), there are cheap ways to test for the presence and quantities of it.
Great Presentation! When you have the sticky ends, how come they are always AATT? Is that due to the restriction enzyme that you used or is there another reason for having the same sequence?
Hi Eitan, The AATT sticky ends are always true for EcoRI which is an example of a conventional restriction enzyme, where the cut site is in the middle of the recognition site. The enzymes we used, BsaI and AarI are type 2 restriction enzymes where the sticky ends can be any 4-piece combination of the 4 base pairs. This is because BsaI and AarI cut outside of the recognition site.
How would the synthase gene be incorporated into the soy bean?
We will transfer our final vector plasmid from E. Coli to an agrobacterium – a type of bacteria that infects plants. From there we will allow it to infect the soybeans and incorporate the engineered plasmid into the soybean genome
Great Presentation!! When you mentioned the sticky ends you said that it was always AATT? Is that due to the restriction enzyme that you used and will it always be like that?
How could the synthase gene be incorporated into the soy bean?
You mentioned that the Golden Gate method is irreversible by the same enzyme, how is this beneficial?
Thanks for asking this! We didn’t really have time to explain that in the video. Because the recognition site is removed when the new plasmid is created, there is no “scar” on the new plasmid. This means we can just slap on the recognition site anywhere onto any sequence we need, without having the recognition site in the end product. Whereas with the reversible conventional enzymes, you would need to conveniently have the recognition site for those already part of the sequence.
If you were to alter yeast and bacteria to have the GDP pathway, what would that affect? Do you think it would have the same exact effect as soybeans?
It would have the same effect, it would just take extra steps to implement the whole pathway.
How were the three different methods used of the successful integration of the E.Coli determined?
There’s varying levels of accuracy for each method and ease of execution:
Sequencing is the most accurate, but it takes the most time to send off and we have to pay so it would be inefficient to use it for every step.
The antibiotic resistance is good to make sure the growths have the plasmid but it’s not guaranteed that all the colonies have the pinene gene and the antibiotic resistance so it’s much lower accuracy, but a good way to track steps.
The gel electrophoresis is a good middle ground because it’s more accurate than the resistance, but it can be done in lab at the time.
What types of flavors and scents are terpenes responsible for?
The point of terpenes is to allow plants to interact with their outside world- it may attract pollinators, deter pests, help plants recover from injuries, or make them tasty or not tasty to predators/things that would spread their seeds. They can be citrusy, earthy, spicy, etc. If you think about the wide variety of smells found in essential oils- those are comprised of lots and lots of terpenes.