Results

Overview

In this section, we will present all the results that are important checkpoints in our experiments. These include successful plasmid construction verification, successful protein expression in E. coli, successful transformation into Syn. PCC 6803, and successful terpene production in Syn. PCC 6803.

Verifying the Construction of terpenes produce circuit in E.coli ///

Colony PCR of E. Coli Single Colonies

Below are the results of the gel electrophoresis of colony PCR of transformed single DH5alpha E. coli colonies with our constructed plasmids (Figures 1-3). The sizes that are same as the expected size are boxed. The constructed plasmids are made from the assembly of the terpene synthase gene segments and our shuttle vector.

Figure 1: The growth of αPS, βPS and LIMS colonies on petri dish

Figure 2: αPS Colony PCR Gel Electrophoresis Results

Figure 3: AgBS, βPS, LIMS, PaFS, PtPS, SaSS Colony PCR Gel Electrophoresis Results

Figure 4: LIS Colony PCR Gel Electrophoresis Results

Figure 5: MsLIMS, LIMS Colony PCR Gel Electrophoresis Results

The correct sizes of all the segments are listed below:

Terpene Synthase Gene	Expected Size (bp)	Results
AgBS	2454	Successful ✔
αPS	1884	Successful ✔
βPS	1881	Successful ✔
LIS	2613	Successful ✔
LIMS	1671	Successful ✔
MsLIMS	1800	Successful ✔
PaFS	1743	Successful ✔
PtPS	1845	Successful ✔
SaSS	1710	Successful ✔

If the sizes are similar to the correct sizes of the fragments, then there is a chance that the transformation is successful, and the plasmid construction is correct. We will send these positive colony PCR samples to sequencing to test whether they are exactly what we want.

The following genes have at least one positive colony PCR sample: AgBS (Figure 3), αPS (Figure 2), βPS (Figure 3), LIS (Figure 4), LIMS (Figure 5), MsLIMS (Figure 5), PaFS (Figure 3), PtPS (Figure 3), SaSS (Figure 3).

Sequencing

Sequencing of the plasmids can test whether our constructed plasmid is exactly the same as what we designed and inserted. Below are the diagrams of the sequencing results, the solid red arrows present at the top of the diagrams mean that the sequencing is correct.

Figure 6: Sequencing result Snapgene diagrams of AgBS, αPS, βPS, LIS, MSLIMS, PaFS, PtPS, SaSS, SaSS-CYP736A167

Terpene Synthase Genes	Results
AgBS	Successful ✔
αPS	Successful ✔
βPS	Successful ✔
LIS	Successful ✔
LIMS	Unsuccessful ×
MsLIMS	Successful ✔
PaFS	Successful ✔
PtPS	Successful ✔
SaSS	Successful ✔
SaSS-CYP736A167	Successful ✔

All of our plasmids have positive sequencing (Figure 6) except for LIMS.

Verifying the expression of terpene synthases in E.coli ///

Through SDS-PAGE, we can verify the expression of all the circuits we constructed in E. coli BL21(DE3) with 37◦C, 0.5 mM IPTG. It is regrettable that most proteins, including AgBS, αPS, and MsLIMS, were not expressed. We think that only some of the proteins were expressed because these conditions and the chassis are not optimal for the expression of all the proteins.

Figure 7: SDS-PAGE protein check gel electrophoresis result of AgBS, SaSS, PaFS, αPS

pET28a-PaFS (Figure 7) and pET28a-SaSS (Figure 7) both have desired protein expression, compared to the control. However, pET28a-AgBS and pET28a-αPS do not have the desired protein expression.

After discussions within our team and with our advisors, we learned that all the terpene synthases came from plants, and there are some scientists [1] that used cyanobacteria as the chassis to produce these terpene synthases. This made us more determined to move on to use cyanobacteria as the chassis of our terpene production.

Successful Transformation into Synechocystis sp. PCC 6803 ///

Colony PCR of Syn. 6803 Single Colonies

Figure 8: Streak monocultures of transformed Synechocystis sp. PCC 6803 with terpene synthase genes AgBS, αPS, βPS, LIS, MsLIMS, PaFS, PtPS, SaSS, SaSS-CYP736A167

Below are the gel electrophoresis pictures of the colony PCR of transformed Syn. PCC 6803 with fully constructed plasmids, which are sequencing positive. The single colonies are shown above. If we see that the gene segment is the correct size, then we know that the plasmid is inside the cyanobacteria, and we can begin culturing them for gas chromatography check. The ones that are boxed are all the right sizes.

Figure 9: Colony PCR of AgBS, βPS, PaFS, PtPS, SaSS in Syn. PCC 6803 monocultures

Figure 10: Colony PCR of αPS, SaS-CYP736A167, MsLIMS, LIS in Syn. PCC 6803 monocultures

The correct sizes of all the segments are listed below:

Terpene Synthase Gene	Expected Size (bp)	Results
AgBS	2454	Successful ✔
αPS	1884	Successful ✔
βPS	1881	Successful ✔
LIS	2613	Unsuccessful ×
MsLIMS	1800	Successful ✔
PaFS	1743	Successful ✔
PtPS	1845	Successful ✔
SaSS	1710	Successful ✔
SaSS-CYP736A167	1503	Successful ✔

The following genes have been successfully transformed into Syn. PCC 6803: AgBS (Figure 9), αPS (Figure 10), βPS (Figure 9), LIS (Figure 10), MsLIMS (Figure 10), PaFS (Figure 9), PtPS (Figure 9), SaSS (Figure 9), SaSS-CYP736A167 (Figure 10).

Gas Chromatography ///

Through the help of our advisors, we tested whether the terpenes are being produced by our Syn. PCC 6803 after the inducer is added using gas chromatography. To see whether the test is successful, we check whether there is a peak at the same place as the model sample in the database. Below are the chromatograms of the products that are correct, being beta pinene and farnesene. We are still in the process of testing the other terpenes.

Figure 9: The GC result of β-pinene and α-Farnesene