Introduction

If you are reading this, I am inclined to assume you already know a bit about synthetic biology (SynBio). By a bit, I mean that you are aware how centred the field is around creating and adapting standardized approaches to solve problems. For example, genetic parts are to synbio as nuts and bolts are to a mechanic.

There are some standards already such as metric and imperial, and they all do a job. However, once you start working with one system, it’s hard to justify working with the other.

I know of labs which still perform restriction-ligation using Type-II enzymes. It’s not terrible, and in fact, yields results. But it begs the question; why? Why is it so hard to adopt new standards? Is synbio evolving too fast? If so, should we put a pause on standardization and focus more on implementation?

There are many questions, many of which I will try to answer in this article. So, if you would please, join me, and embark on this journey of meta-engineering synthetic biology.

What do we know already?

To find out what makes a standard, it is important to understand what we know already. On whose shoulders does synbio stand? Moreover, what kind of standardization exists? What kinds does the field really need? For this article, I will focus on assessing needs for wet lab practices. However, I will touch on the needs of data reporting in a future article.

DNA standardization

In order to treat sequences of DNA as “mechanical” parts, we must formalize how we describe and use them in the laboratory. A prominent example of this is the BioBrick standard of 2002 (strangely enough, the year in which I was born). This standard allowed researchers to use a common language to describe and use DNA parts. Moreover, the standard allowed for idempotent transformation of DNA, such that parts may be stitched together without losing their function.

In the years following the BioBrick standard, many other standards have been proposed. One of these is MoClo; it is built upon Golden Gate Cloning. This is the method which my iGEM teams used in 2022 and 2023. More on this later.

These processes are very useful. However, they are quite complex to use, as all source DNA must be “domesticated” to fit the standard. This becomes a particular problem when performing functional metagenomics. Untargeted modification to DNA can arise a multitude of problems, such as premature termination of sequences – introducing loss-of-function mutations. Moreover, genes might need to be domesticated in multiple different ways to allow for a truly modular approach.

Imagine a enzyme which can modify the terminal ends of any DNA with a specific sequence. Would that solve the problem of prerequisite domestication?

WRITE A PART ABOUT DNA BEING REPORTED WEIRDLY. NOT IN FULL CONTIGUOUS SEQs AND NOTHING DOMESTICATED As of writing this, DNA has been…

Plasmid assembly abstractions

To addition to MoClo, plasmid collections such as pJUMP and pSEVA provide their own set of restriction enzyme protocols and asymmetric overhangs which make it very easy to plug-and-play once genes are domesticated. ADD LINKS

The interesting thing about assembly abstractions is that, while much research has gone into the efficiency/reliability of four-base overhang combinations and enzyme choice, I find that their strength lies in their intrinsic genetic parts. Plasmids with better or a broader range of ORIs, antibiotic resistances, and even screening reporters will be chosen over more restrictive models.

Here are a few points I would like to share about assembly abstractions.

• Blue/white screening shouldn’t be the status quo anymore. We have far better ways to screen using small chromoproteins and constitutive promoters. Why do we still require using IPTG and X-Gal on every plate?
• Modularity is great. We are already assembling plasmids; assemble some more! Breaking down vectors into parts (ORI, AbR, screening reporter, etc.) gives more freedom to those working with unorthodox chassis.
• Along the lines of the previous, we need to start working on making plasmids for non-model organisms. Modularity would help with that – but what about using learning from functional metagenomics to produce untargeted promoter libraries? Sure, it’s not hard-hitting research; but it is what we need to unlock the potential of other organisms.

Chassis engineering

Chassis engineering is a field which is still in its infancy. However, it is becoming increasingly important as we move towards more complex systems.

Why is chassis engineering so important? Since I heard of it years ago, I have been fascinated by bioorthogonal chemistry. This is the study of chemical reactions which can occur in a biological system without interfering with natural metabolism. This is exemplified by the recent work of Click Chemistry, which has been demonstratably used in living systems. Now, synthetic biology inherently involves the use of metabolites within the cell – so it is difficult to say that it’s truly orthogonal. However, we can engineer chassis to withstand diversion of its carbon flux, or even adapt to it – as though it would give its own metabolites for the cause of humans. Perhaps: biorouting protocols?

In terms of standardizing chassis engineering, there is no precedent. For now…

A common method for engineering cell lines is by some mechanism of inserting DNA into the genome by allowing it to recombinate with plasmid DNA. For example, through phage engineering or using large flanking regions that are homologous to the target locus. An example of the latter is commonly found when engineering Agrobacteria tumefaciens to recombine with plants as a gene delivery vector. In order for this to occur, the target DNA has to be incorporated to the crown gall region of the Agrobacterium genome. ADD SOURCE HERE

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