Engineering Success

Introduction

When planning out and finalising our project idea before summer, everyone in the team had aimed to undergo multiple design cycles. We were aspiring to test many different approaches; including: pumps, feedback systems to shut off enzyme production, degraders to stop further production or even using enzymes to detoxify the products which could be converted back after extraction, just to name a few. By the end of the summer, we aspired to have an optimised and innovative solution for the production of perillyl alcohol in E. coli using information gathered from experiments and modelling.

However, with substantial delays in the delivery of our parts as well as some large changes to the experiment that had to be made part way through (which will be discussed later), we had a much tighter time frame to run it through numerous cycles than we had hoped. Despite this, taking a step back to optimise or redesign our experiments entirely was vital in reaching our desired end goal before the iGem deadlines.

Changing pJBEI-6410+ Plasmid Assembly Method

When designing the feedback system of our project, pJBEI-6410 only contains a small ~50bp tet promoter sequence that will respond to the tet produced by pJBEI-6411 to downregulate the expression of downstream enzymes. Because it is such a short sequence, we intended to add the sequence onto the end of primers and clone it in by an overlap extension PCR instead of ordering a large gibson fragment that is mostly backbone.

We designed the complementary primers around specific restriction sites and, after they eventually arrived, we had early success in the production of A and B fragments, joining the A and B fragments together and cutting open the plasmid. Despite this, after weeks of repeats and slight alterations to the procedure, we still couldn’t produce cells containing the modified plasmid. Most of our samples didn’t grow cells on ampicillin plates, and the few colonies that did grow failed our colony PCR tests (an example gel is shown in Figure 2).

Using HP20 Resin to Isolate Our Products From Liquid Culture

With perillyl alcohol and limonene not having a unique absorbance signal to identify them with, our chosen method of analysing their presence was using gas chromatography-mass spectrometry (GCMS). After growing a liquid culture and homogenising the cells, all of the leftover cellular components in the sample can create a lot of background noise on the GCMS trace and make our products harder to extract. Because of these reasons, and hearing the idea from a chemistry professor, we aimed to use HP20 resin during the extraction. It has been used to isolate terpenes of similar structure to PA and limonene in previous research so, with only one functional group atom difference, we believed that it could be equally very effective (Ignea et al., 2019).

Regrettably, however, as shown in Figure 3, the addition of HP20 resin removes the PA peak at ~6 minutes entirely and isn’t especially effective at removing impurities either. Because of this, we obviously cannot use the resin during extraction. During our second run of GCMS (being carried out post wiki freeze deadline) we are however using an internal standard that, despite not removing any impurities, should make it possible to more accurately assess the amount of PA/limonene produced. Any updates to our progress using the internal standard will be shown on the results page.

Modelling

During our project the modelling team helped us to plan some of our experiments, including the IPTG induction assays that allowed us to express the plasmid at different levels to see the effect of our pathway and its products on growth. The act of balancing production of PA/limonene against the growth of our cell is something that we were considering a lot in the creation of our project. More info on the modelling page.

References