Engineering Success

Overview

We always follow the Design-Build-Test-Learn Cycles to move the project forward. This year's project can be roughly divided into three cycles. In the first cycle, we finished building and testing the terpene synthesis circuit in an E. coli chassis. In the second cycle, we achieved to express iteratively terpene synthesis circuit in cyanobacteria, and in the third cycle, we designed kinds of bioreactor to match the engineered cyanobacteria.

Figure 1: Engineering cycle diagram of our project

Cycle 1: Terpenes synthesis circuit construction in E.coli ///

Design

Due to the fast growth rate of E. coli and the ease of molecular cloning, we initially decided to construct and validate all the terpene production circuit in E. coli. Through gene synthesis, we obtained the target gene, assembled it into plasmid pJA and transformed it into E.coli DH5α.Then, we extracted the plasmid from DH5α and tried to transform and express the terpene synthases in E.coli BL21(DE3).

Figure 2: Snapgene diagrams of AgBS, αPS, βPS, LIS, MsLIMS, PaFS, PtPS, SaSS, SaSS-CYP736A167

Build

  1. Plasmid Extraction 0802
  2. Normal PCR 0804
  3. Gel Electrophoresis 0803
  4. Assembly 0805
  5. E. coli Transformation 0806
  6. Colony PCR 0807a & 0807b

Figure 3: Colony PCR results for all our samples

Test

0.5mM IPTG is added to BL21(DE3) to induce the terpene synthases expression. Through SDS-PAGE, we tested less than only two out of the five tested terpene synthases were be expressed in BL21(DE3).
Figure 4: SDS-PAGE protein check gel electrophoresis result of AgBS, SaSS, PaFS, αPS
Figure 5: Gas chromatography results of terpene production by E.coli(The results of α-Pinene is shown here as an example).

None of the gas chromatography of terpene production by BL21(DE3) have the desired results. As can be seen in Figure 4, the places that have high peaks in the standard product are not the same as the places in our α-pinene.

Learn

Because less than half of the samples we tested expressed terpene synthases and no samples successfully synthesized terpenes. We needed to find a more efficient way to produce terpenes. Through discussions with our advisorss, we learned that E. coli contains very few of the key precursors for terpene synthesis and is not an ideal chassis for expressing terpenes. Cyanobacteria, although genetically complex to manipulate, are undoubtedly much better at expressing terpene synthases. Therefore, we decided to move on to use cyanobacteria, Synechocystis sp. PCC 6803, as our chassis in the next steps.

Cycle 2: Terpenes synthesis circuit construction in Cyanobacteria ///

Design

We use electroporation to transform the plasmids we constructed in DH5α E. coli into Synechocystis sp. PCC 6803. After being cultured and induced for a few weeks we add dodecane into the cultures to extract the terpenes, and test whether the terpenes are being produced through gas chromatography.

Build

  1. Cyanobacteria Electroporation Transformation 0809
  2. Streaking Cultivation 0810
  3. Gel Electrophoresis 0803
    4. Figure 6: Streak monocultures of transformed Synechocystis sp. PCC 6803 with terpene synthases genes AgBS, αPS, βPS, LIS, MsLIMS, PaFS, PtPS, SaSS, SaSS-CYP736A167
  4. Cyanobacteria Colony PCR 0811
    5. Figure 7: Colony PCR of AgBS, βPS, PaFS, PtPS, SaSS in Syn. PCC 6803 monocultures
    Figure 8: Colony PCR of αPS, SaS-CYP736A167, MsLIMS, LIS in Syn. PCC 6803 monocultures
  5. Culture of Monocultures in BG-11 Culture Medium in Erlenmeyer Flask for 2-3 Weeks at 30◦C in Light Bioreactor

Test

Through gas chromatography, we can see whether the products that our cyanobacteria produced are the same as what we engineered it to produce. Both PaFS and βPS were successful in producing the wanted terpenes. The others are still in the process of testing.
Figure 9: picture of induced Syn. PCC 6803 we engineered. The transparent layer at the top is dodecane, which can dissolve the terpene molecules, making them easier to test.
Figure 10: Gas Chromatography results confirming production of beta pinene and farnasene

Learn

Though we are able to produce the scent molecules, we are still unable to directly fulfill the purpose of our project, which is to relieve stress through aromatherapy, and to bring our project into people's daily lives. Part of it is because of the safety restrictions of the competition, but even more so is that we do not have a way to do it. From our HPs at our schools, we further confirmed that students face a lot of stress and would really like to have our prospective product in their daily lives. Therefore, we decided to build different bioreactors.

Cycle 3: Hardware Design ///

From the previous two experimental cycles, we have successfully engineered cyanobacteria to produce scents. Our next main cycle will be about building hardware that the cyanobacteria can thrive in. These hardware will be designed so that it can bring greenness and sustainability into the daily lives of people, and lead people out of stress. In this section, we also experienced 3 engineering cycles to get our bioreactor designs to be able used in multiple life contexts.

Module 1

Our project emphasizes greenness and sustainability, with a focus on cyanobacteria, a unique organism independent of organic carbon sources and capable of photosynthesis. To start with designing hardware, we tested cyanobacteria cultivation in common household items like fish tanks or jars, considering factors such as climate, temperature, light, and humidity, based on the different location and family conditions of our team members.

Module 2

Our first trial in tanks and jars showed cyanobacteria growth relies on a stable environment, which prompts us to create a device that helps with its cultivation and use. The desired apparatus is supposed to maintain a stable environment, provide artificial light and oxygen sources, and allow for proper circulation. A bioreactor, or an apparatus with pumps and transparent tubes which cyanobacteria circulates within may be the solution to maintenance of a stable environment.

Module 3

In order to produce a new version of the bioreactor that blends well into daily use, our team designed two serials of the bioreactor that have a close resemblance to common large and small items in everyday life. The large bioreactor imitates flowing water sceneries that are common to public facilities and offices. The small bioreactor consists of a wide array of daily items that is correlated with scent. The combination of bioreactors, large and small, provides solutions to anxiety and stress by surrounding us with fragrances.

For more information, please visit Hardware.