The Inscripta Blog

February 15, 2022

Introducing the Onyx® Iterative Genome Engineering workflow for rapid strain improvement

Inscripta recently launched a new feature to expand what you can do with the Onyx platform – Iterative Genome Engineering, which enables introduction of additional edits into previously engineered strains. The Onyx Iterative Genome Engineering workflow makes it easy to move from one engineering round to the next, cutting down the time between editing cycles to just one week.

What this means for you is – reaching your strain improvement goals faster. With this workflow, you can take up to 24 strains to the next editing round, either as single isolates or in a pooled format. We provide validated protocols and a kit to (for E. coli strains) ensure each engineering cycle is successful and new edits are incorporated at a high rate. Let’s take a closer look at what you can do with Iterative Genome Engineering and how it can save months of development time.

What can you do with Iterative Genome Engineering?

Iterative Genome Engineering can be applied to a broad array of applications from protein and metabolic engineering to strain optimization. In a recent webinar, Eric Abbate, Director of Analytical Biochemistry from our Applications Development team, describes the Iterative Genome Engineering workflow for protein engineering and improving protein production.

In the first case study, our scientists engineered the green fluorescent protein (GFP) gene in the E. coli genome and identified two novel variants with a strong spectral shift. In the second application, we used a whole-genome approach to improve the production of an important bioindustrial enzyme in S. cerevisiae. With 3 engineering cycles, we were able to obtain nearly a 4-fold improvement in less than 6 months. You can watch the webinar or read the app note to learn more.

How does Iterative Genome Engineering reduce hands-on time?

To answer this question, we have to understand the steps required to take the strain from one engineering cycle to the next. During the engineering run, the cells are transformed with editing plasmids. After phenotyping and selecting the strains you want to take forward to the next engineering cycle, those plasmids have to be cured from the cells prior to transforming them with new Design DNA.

Curing may seem like a standard lab procedure at first, but it could turn out to be harder and take a lot more time than initially expected. S. cerevisiae cells grown without the antibiotic selection required to maintain the plasmids will gradually lose them over the course of serial growths and dilutions. This process is called passive curing, and we provide a protocol that describes in detail how to perform it with up to 24 variants. For E. coli, it’s a little more difficult to rid the cells of the plasmids, making it necessary to go through what is called ‘active curing’. For this purpose, we have developed an Onyx® E. coli Curing Kit that enables curing using the Onyx instrument, with minimal hands-on time.

The most important step of curing, however, is the quality control (QC) process to ensure that the plasmids have been completely eliminated from all cells. If curing is not performed properly, the results of the next engineering experiment will be compromised. Our validated QC protocols help you assess the results of curing and be confident that your next engineering run will be successful.

After curing the plasmids, you will the edited strain’s genome to design new edit libraries. To do that, we have designed a simple tool called Caber that enables you to update and import the genome of the strain you want to use for the next round of editing, without having to spend extra time and resources on sequencing it.

The take-home message

Regardless of the application you are pursuing – whether it’s protein or metabolic engineering, protein production, or engineering strains with unique properties like tolerance to toxic compounds – reducing the time of each engineering cycle and rapidly accumulating beneficial edits is the key to success. With the Onyx Iterative Genome Engineering, the process is optimized and streamlined to ensure high success rate of genome engineering experiments and minimum time between cycles. If you want to learn more about how you can accelerate strain improvement, go to our website or reach out to us to talk about your projects.