Written by Hannah Edstrom
Here we continue our study of this week’s review paper: The Synthetic Biology Toolkit for Photosynthetic Microorganisms. Last time we introduced the concept of harnessing the power of photosynthesis to make natural products. An engineered photosynthetic organism would use light and carbon dioxide, rather than glucose or another carbon source. Here, we can dig into the details a bit more: What kinds of tools do you need to engineer such a microorganism?
But first – how do you pick that microorganism? What makes it an ideal host?
Picking the Host Microorganism
If you want to engineer a microbe to generate some natural product of interest, you’re going to have to mess around with a lot of DNA. So who’s DNA?
The host organism holds all the machinery you’re looking for. There are some other genes you will need – genes encoding enzymes that push each step of some metabolic pathway. That pathway will eventually result in the synthesis of your product. Adding DNA into your host organism is done via transformation.
This host, your ‘chassis strain,’ should grow quickly, transform naturally, and have a small, easily managed genome. Bonus points if it’s resilient – that is, if it can readily adjust when the environment changes (something like pH, temperature, or salinity.)
In the world of prokaryotes, a cyanobacterium does just the trick. In eukaryotes, the race is still on, but at present, it appears to be neck-to-neck between Chlamydomonas and Phaeodactylum. Molecular biology tools exist in all of these.
1. Some Sequences of DNA
You will need various genetic parts in your toolbox. This includes:
Let’s review the Central Dogma. DNA is transcribed into RNA which is translated into proteins.
That means that a gene in DNA gets transcribed into messenger RNA (mRNA) which gets translated into a protein. In the context of synthetic biology, that protein is an enzyme that catalyzes a reaction in your metabolic pathway.
A complete toolkit will have both constitutive and inducible promoters available for use. Promoters are genetic sequences that tell the RNA polymerase where to bind. It is a flag on the genome that says “Read here!” – and some of them are more obnoxious than others. The more obnoxious the promoter, the more that gene is transcribed, so the more of that enzyme ends up in the cell.
Strong and weak promoters are both important when engineering a new pathway. Both of them exist for all three host strains here. Since the promoter ultimately controls how much of the enzyme is in the cell, you will want to fine-tune its strength at each step. The cell’s metabolism should be carefully balanced; using the strongest promoters every step of the way is more likely to make the cell sick than to result in super-production.
Constitutive promoters are “on” all the time. Sometimes, that’s not ideal. Inducible promoters can be turned on or off, allowing the cell to maintain some level of homeostasis. Light is a common inducer for promoters in our chassis strains (after all, they are photosynthetic). Turn on the light, turn on your promoter, begin synthesizing that gene. Turn it off, and the promoter stops signaling for transcription. Other inducers include salt (for Chlamydomonas) and metal ions (for cyanobacteria).
Posttranscriptional Control Tools
You can also control gene expression after transcription. RNA sensor molecules and manmade microRNA fragments can bind to the mRNA. These kind of tools interrupt transcription, giving you further control over how much expression occurs for a gene.
Once you’ve engineered into your host, you’ll want some sort of marker to (1) check that your genetic edit is actually there and (2) kinda low key force the chassis to keep your DNA. Cells are actually pretty good at spitting out unwanted DNA. If your pathway involves some weird or metabolically excessive steps, you will need to cajole your microbe into keeping it.
This is done with selectable markers. Antibiotics are a perennial favorite, including kanamycin, spectinomycin, chloramphenicol . . .
This works by adding a gene for antibiotic resistance (along with whatever else you’re adding). After transformation (that is, when you give the DNA off to your host), you grow it in media containing the antibiotic.
If the strain behaves itself and accepts your DNA, it will survive. Now you know it carries the antibiotic resistance marker, and therefore, also your DNA. If it failed transformation or kicked the new DNA out, it will perish when exposed to the antibiotic.
That’s one way to teach your microbes to listen.
Finally, your genetic toolbox should have a couple reporter genes to work with. The textbook favorite here is GFP. Attach a gene for Green Fluorescent Protein to your DNA insert, and if you succeed, your microbe will glow.
Even better, it will glow with an intensity determined by how strong your promoter is, allowing you to compare expression rates.
Various fluorescent proteins and luciferases can be used here. In cyanobacteria, inducible promoters are used as well.
2. Various Vectors to Put the DNA to Work
Ok. You’ve got a whole bunch of DNA you want to offer your host. There’s your gene coding for an enzyme on your metabolic pathway. There’s a promoter carefully picked for strength and inducibility. You added a selection marker, and maybe a reporter gene and/or posttranscriptional regulator sequence as well.
Now, it needs to be actually inserted into the host. There are two ways to insert DNA into a microbe. You can integrate it into the genome, or you can keep it on a plasmid that stays floating around in the cell. These free-floating plasmids are referred to as replicating plasmids. If a plasmid contains DNA that will integrate into the genome, if is an integrative plasmid.
I’ve thrown in the word plasmid. The ‘vector’ – the vehicle for transporting DNA – is usually a plasmid, which is a circular piece of DNA containing all the your sequences. Transcription occurs directly off of the vector in replicating plasmids, and the genes in integrative plasmids are transcribed after assimilating into the genome.
What’s more, if your plasmid is replicating, it can either be a “high copy” plasmid or a “low copy” one. High copy plasmids exist in very high numbers inside the cell, which means that there will be a larger pool of your enzymes floating around. Low copy plasmids are fewer and thus, provide lower enzyme concentrations. Just as with promoter strength, having high copy plasmids isn’t always ideal, and many experiments use plasmids of different copy numbers. This allows you to optimize the exact amount you need for the max possible titer.
Titer – how much of your desired product is synthesized by your microbe
Vectors like these exist for cyanobacteria and our eukaryotic hosts. Integrative plasmids are usually preferred for long-term engineering projects; they tend to be more stable. However, it is here that cyanobacteria present a special problem: each cell carries multiple genomes! It is difficult to tell if an integration was successful in each copy of the genome, making replicating plasmid builds much more feasible here – at least for now.
3. Tools to Edit the Genome Directly
The modern molecular biology toolkit is incomplete without some direct-genome editing tools to work with.
You’ve probably already heard of methods like this. Crispr Cas9 is one of them.
Crispr Cas9 allows you to delete and insert at specific locations in the genome. Cas9 is a type of protein called an endonuclease, which can cut DNA from the inside. We can then use this cut site to make changes to the genome. Here, for example, you might use it to cut the genome and then directly insert your pathway’s gene.
Other kinds of genome-cutting tools include Zinc-Finger Nucleases and TALENS. Only Crispr Cas9 is listed as a genome editor for cyanobacteria, but all three of these methods have been used in eukaryotic algae. ZFN and TALENS are typically more expensive than the cheap new Crispr system.
Regardless of the scissor system, when DNA breaks, it can repair in one of two ways. Keeping in mind that ‘homologous’ is a fancy way of saying ‘the same,’ these two repair methods are ‘nonhomologous end joining’ and ‘homologous recombination.’
Nonhomologous end joining just means that the DNA is broken, and two ends of DNA – which are not the same – bind together anyway to repair the break. That’s nonspecific, can easily result in mistakes, and may lead to some unexpected problems down the road.
Homologous recombination is often preferred, since it uses genetic sequences that match DNA on either end of the break. For example, if I have the sequence of the gene I want to insert, I can extend that sequence with nucleotides that match the host’s DNA on both open ends. That way, when the host genome repairs from the cut, DNA sequences will match up and bring the new gene along with them. That is a very specific repair mechanism – one I have a lot more control over.
If you want to reduce nonhomologous end joining and improve your chances that homologous recombination will work – and are working with Chlamydomonas – you’re in luck. Researchers recently discovered that if you knock out a single particular gene, you decrease nonhomologous end joining and improve your chances of a successful transformation.
So that’s one final tool to throw in the box.
We’ve gone over a lot of information today! Let’s conclude by putting this all in context. These are synthetic biology tools, useful when you want to metabolically engineer an organism for the synthesis of some natural product.
Synthetic biology – a field or redesigning or modifying natural systems, like a cell
Metabolic engineering – modifying a cell by modifying its metabolic pathways
Every cell has a metabolism, including pathways to thousands of molecules it can produce. Our desired product may be native to that metabolism, or it may be foreign; we can insert genes for enzymes not native to a cell’s metabolism, or we can tinker with the genes already there (if they code for enzymes related to our pathway).
Natural product – the desired creation, the molecule you want your microbe to synthesize for you
Maybe you have a molecule that can treat cancer, nourish crops, replace plastic, beautify your face, or act as a fuel. If there is a pathway from glucose to that natural product – if enzymes exist for each chemical step – you can insert the genes for that pathway into a microbe.
Of course, as we advance our synthetic biology toolkit for cyanboacteria and other algae, we get closer to making natural products out of carbon dioxide instead of glucose
The genetic tools needed for this kind of engineering is many and diverse. But as this review details, we are already well on our way.
Thanks for stopping by and thanks for reading!