Science
Building Your Own Plasmid
Recombinant DNA has been one of the fundamental breakthroughs within Synthetic Biology of the last century. It offered us a means to insert DNA fragment from an external source into the genetic material of an organism, allowing it to obtain enhanced and desired characteristics in living organisms
It’s crazy if you think about it… how we’re able to take a piece of genetic code from an entirely different organism and with it, manipulate the genome of a living creature.
How does this work exactly? Let’s break it down!
Plasmids
Plasmids are circular pieces of DNA that replicate independently from the host’s chromosomal DNA. This references the concept of “modularity”, a key engineering principle of synthetic biology. We often refer to plasmids as vectors, because they act as “vehicles” for inserting isolated DNA into living cells.
All plasmids consist of the same basic components:
- Origin of Replication
- Restriction Sites
- Gene Insert
- Selector Marker
Origin of Replication (ORI)
An ORI is a specific sequence of DNA that initiates replication within a plasmid. It does this, by recruiting plasmid-encoded transcriptional proteins which recognise specific DNA sequences & determines it as the ORI.
Origins of replication are typically either relaxed or stringent. Relaxed ORIs occur freely within the cytoplasm, and often yield high copy numbers of plasmids. Stringent ORIs, however, rely on the replication machinery of the host cell’s (synthesised by the bacterial chromosomes) and often yield low copy numbers.
Restriction Sites & Multiple Cloning Sites
Restriction enzymes (endonucleases) are like killer robots, programmed with a single target in mind — to cleave a complementary 4–8p sequence, called a restriction site. They make a cut at that site, through two strands of DNA, typically leaving a 2–4 nucleotide single-stranded overhand in its wake.
Oftentimes, within commercially-engineered plasmids, restriction sites are grouped together in Multiple Cloning Sites (MCS). A MCS is a short segment of DNA, containing up to 20 restriction sites, allowing for the insertion of foreign DNA without disrupting the rest of the plasmid.
When selecting a restriction site, we should take into account the number of cut sites. Cut sites are segments along the genome restriction enzymes will bind to & cleave. Ideally, we’d select a restriction site with only 1 cut site. If the restriction enzyme cleaves at >1 site, we’d get multiple plasmid fragments and the plasmid itself, will likely be of no use.
Gene Insert
The gene of interest is what creates new functionality within the plasmid, and is the reason for the experiment in the first place. The gene of interest can be virtually anything, from GFP that causes cells to fluoresce in green to Bt which expresses insecticidal protein in its cells!
After the same aforementioned restriction enzyme isolates the gene, it’s inserted into the plasmid using DNA ligase. Here, DNA ligase acts as a glue to allow for “annealing” — the formation of hydrogen bonds between complementary sequences on single strands — sealing the gene into the plasmid.
Selection Markers
Once the gene-of-interest has been inserted into the plasmid, it’s now used as a vector, to be inserted into a living cell! This often comes in the form of E. coli, a well-characterised & simple bacterial cell.
The problem is, successful plasmid transformation (the uptake of the plasmid) is notoriously low, with only 1 out of 10, 000 cells succeeding on average. To quickly filter through these plasmids, scientists ingeniously engineered plasmids to include selection markers. Selection markers allow scientists to identify bacterial cells that have been successfully transformed, while the rest are killed off in the experiment.
Antibiotic Resistance
For example, researchers often engineer plasmid backbones to contain an antibiotic-resistant gene. They then plate the bacterial cells on a growth medium containing antibiotics — agents that either kill bacteria or dramatically inhibit their growth (e.g. Ampicillin). If the bacterial cell contains the ampicillin-resistant gene, it will express an enzyme called b-lactamase. B-lactamase acts like a “defense system”, catalysing the hydrolysis (removal of a water molecule) and subsequent “destruction” of ampicillin in the surrounding area.
Auxtrophy
In eukaryotic host cells, this gene is often replaced with an auxotrophic selection marker. Auxtrophy is an inability of an organism to synthesise a particular organic compound required for its growth.
Scientists therefore engineer the host cell, such that’s no longer able to produce essential organic compounds. These abilities are then conferred to the plasmid, meaning that only successfully transformed host cells will be able to survive. For instance, TRP1 is often used as a selection marker for the amino acid tryptophan. It encodes an enzyme, trpD, that catalyses the third step in tryptophan biosynthesis, enabling the cell to produce tryptophan and survive!
Creating a Plasmid from Scratch
Step 1: Importing the Plasmid Backbone from addgene into Benchling
What’s Benchling? It’s essentially the fullstack software of genetic engineering rom project documentation and data acquisition to sequence design, sample management, process management, and reporting!
Meanwhile, addgene is a free plasmid repository - giving you access to thousands of real plasmid backbones which you can import and use in your experiments.
For this experiment, I used the empty backbone pXP420, which is really simple vector to start with!
There are quite a few terms here, so let’s make it a little more comprehensible!
Step 2: Picking a suitable restriction site
We’d ideally pick a restriction site within a multiple cloning site, with 1 cut site. I chose Sac I because it fit this very rudimentary criteria, but in reality, you often pick specific restriction sites based on price, availability of restriction enzymes, etc.
Step 3: Inserting the TEF 1 promoter
These plasmids are often created in the lab, using an assembly procedure called the Gibson Assembly. It’s essentially a reaction that joins multiple fragments of DNA in a single, isothermal reaction.
What happens in practice, is that during the Gibson Assembly process, DNA at the restriction site will simply be “eaten away”. To simulate this, we delete the restriction site of Sac I, replacing it with the TEF1 promoter, and annotating it as such.
Step 4: Inserting the Kozak Sequence
The Kozak sequence (GCCGCCACCAUGG) functions as the translation initiation site in most eukaryotic mRNA transcription strands. We insert this directly after the promoter.
Step 5: Inserting the “gene of interest”
After copying its DNA from an external repository, the gene-of-interest can now be inserted directly after the Kozak sequence. Once this is done, we now need to end the processes of transcription & translation.
Step 6: Inserting a “stop” Codon
Following this, we insert & paste in the “stop codon”, a tri-nucelotide sequence (e.g. TGA) which signals the termination of the translation of the current protein.
Step 7: Inserting the CYC1 terminator
We then copy the CYC1 terminator sequence already present in the plasmid, and paste it directly after the “stop” codon. And voila.. we’re done!
And we’re done!
Once the plasmid has been constructed, it will be transformed into correspondent bacterial cells, where it can later be replicated on a much larger scale.
View my finished project here!
And here’s a really awesome tutorial if you’d like a detailed walkthrough of how to implement this in Benchling!
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Hey, I’m Sarrah Rose! A 17 year old deeply passionate in utilising Synthetic Biology & Artificial Intelligence to solve major problems in the world today. If you enjoyed this article or would just like to chat, I’d love to hear from you:
