There are multiple codons that code for a single amino acid. Every organism has certain tRNAs associated with codons that will be more abundant than others. If an organism runs into a codon that it does not commonly see and has a low abundance of the associated tRNA then it can cause the production of the associated protein to stall. This is where codon optimization comes in. Codon optimization takes a DNA sequence and converts it into a sequence that contains codons that would be more commonly found in the host organism.
I chose to optimize the codon sequence for yeast (Saccharomyces cerevisiae) as this is a model microorganism used for synthetic biology and I would first just like to test if this would work to produce fungal luciferase.
The DNA sequence can now be sent to a DNA synthesis company such as, Twist Biosciences. Twist can provide either DNA fragments or clonal genes that already have the DNA sequence of interest inserted into a vector. If receiving DNA fragments instead of clonal genes, first the fragments will have to be inserted into a vector. Once the fragments have been inserted into a vector, or if clonal genes were ordered instead, this can then be introduced into an organism. For yeast a heat shock or electroporation are used to introduce the DNA into the cells. Once the yeast cells have successfully taken up the DNA, the cells will then proceed to transcribe the DNA into RNA and then translate that RNA into the protein of interest.
How does it work in nature/biological systems?
Describe how a single gene codes for multiple proteins at the transcriptional level.
In eukaryotic systems a single gene can code for multiple proteins because eukaryotic genes contain exons and introns. The exons are the segments of the gene that will end up in the final mRNA product and encode the final protein. When mRNA is being processed the exons can be spliced together in different combinations creating different proteins.
Try aligning the DNA sequence, the transcribed RNA, and also the resulting translated Protein!!!
Prepare a Twist DNA Synthesis Order
Fully annotated Benchling insert fragment
Twist cloning vector
DNA Read/Write/Edit
DNA Read
(i) What DNA would you want to sequence (e.g., read) and why?
I would want to sequence the DNA of all known bioluminescent fungi just to see all the different variations of this system that nature has come up with. Then compare how those differences impact the intensity of the glow or maybe certain variations would work better in certain cirumstances/organisms.
(ii) What technology or technologies would you use to perform sequencing on your DNA and why?
I would want to use use nanopore sequencing technology because it is a relatively low-cost sequencing method. Nanopore sequencing is a third-generation sequencing method because it can read long single DNA molecules directly in real time. The input for nanopore sequencing is typically DNA, RNA, amplicons, or cDNA. First the DNA/RNA is extracted then sequencing adapters are attached to the ends of the strands. The sequencing adapters are oligonucleotides that are loaded with a motor protein. The motor protein associates with the nanopore in the flow cell and controls the DNA or RNA strand movement through the nanopores at a defined speed. Samples are then ready to be loaded and sequenced.
The flow cells used for sequencing the samples contain ion-permeable nanopores embedded in an electrically-resistant membrane enabling an ionic current to pass through the nanopore when a voltage is applied across the membrane. This creates a measurable current that is disrupted when a strand of DNA or RNA passes through the nanopore. The disruption of current is measured and is used to identify the bases passing through the nanopore. The disruption produces a characteristic ‘squiggle’. The squiggle is then decoded using basecalling algorithms to determine the DNA or RNA sequence in real time.
DNA Write
(i) What DNA would you want to synthesize (e.g., write) and why?
I would synthesize the cluster of genes that are involved in the bioluminescence pathway in the fungus Neonothopanus nambi. There are four genes involved in the autonomous biolumiscent pathway in N. nambi. These are hispidin synthase (HispS), hispidin-3-hydroxylase (H3H), luciferase (Luz), and caffeylpyruvate hydrolase (CPH). Here are their associated genetic sequences:
(ii) What technology or technologies would you use to perform this DNA synthesis and why?
I would use chip-based oligonucleotide synthesis followed by assembly because it enables parallel production of many DNA fragments at relatively low cost. Here are the steps for chip-based oligonucleotide synthesis:
Coupling with phosphoramidite - a protected phosphoramidite is added to the unprotected 5’ OH end of the DNA strand
Capping unreacted sites - unreacted 5’ OH are acetylated to prevent further chain extension
Oxidation - oxidation of phosphite triester to phosphate using aqueous iodine
Deprotection - acid catalyzed removal of protective group to allow for subsequent base addition
Accuracy is the biggest technical limitation. Each base addition has a small failure probability. This means only shorter maximum reliable length DNA can be synthesized. Speed can also be a limitation as the chemistry is cycle-based, one nucleotide added per cycle, so the physical synthesis time scales with sequence length no matter how many sequences are on the chip. You can make millions at once, but you cannot make any single one faster than the chemistry allows.
DNA Edit
(i) What DNA would you want to edit and why?
I would want to edit the DNA of various organisms so that they would become bioluminescent. I would start with microorganisms such as E. coli and yeast. Then I would like to move to plants, I have not thought about modifying any other eukaryotic organisms beyond that. My main interest is just to see if this would work well in various organisms to produce autonomous bioluminescence.
(ii) What technology or technologies would you use to perform these DNA edits and why?
I would use CRISPR-Cas9 to make the edits. CRISPR-Cas9 works across kingdoms and it enables precise editing and insertion of DNA. CRISPR-Cas9 has the ability to find and cut specific DNA targets based on the sequence of its guide RNA. The guide RNA can be designed to be complementary to a particular target sequence in the genome. Cas9 will search the whole genome, eventually finding its target site and making a double-stranded break. The double-stranded break can then be repaired through homology-directed repair allowing for the donor DNA to be incorporated into the host organisms genome.
To prepare to edit with CRISPR-Cas9 the first step is to design a donor DNA template flanked by homology arms matching the target locus and guide RNA complementary to the region. Plasmids would then need to be constructed to express Cas9 and the guide RNA. All in all, the inputs required for the editing would include the Cas9 nuclease, guide RNA, donor DNA template, plasmids, primers, and the host cells to be edited. Additional components such as enzymes for DNA assembly and transformation or transfection reagents would also be required to introduce the editing system into the cells.
One limitation of CRISPR-Cas9 editing is the possibility of off-target effects. This is where Cas9 cuts at sites that are similar but not identical to the target sequence and could induce unintended mutations. Another limitation is that cells prefer to do non-homologous end joining over homology-directed repair, which can introduce insertions or deletions. The CRISPR-Cas9 editing system is also limited by the delivery problem, which involves getting the CRISPR-Cas9 system to the target cells. Some organisms or cell types are more difficult to transform or transfect, which can reduce the number of successfully edited cells.
