Parts

During our iGEM journey, our team successfully designed and created 12 genetic parts specifically tailored for Yarrowia lipolytica. These parts are essential for our project, as they are customized to work seamlessly with this yeast strain, allowing us to advance our research and experiments effectively. The parts were designed and codon-optimized in SnapGene to ensure proper transcription in Y. lipolytica. To visit our parts page, click here

Primer Design

Primer design is an essential step for any molecular biology. A primer is a short single-stranded nucleic acid that aids in the initiation of DNA synthesis. A synthetic primer can be referred to as an oligo, short for oligonucleotide.

We had to use primers in order to do PCR Amplification, Integration check PCR and sequencing. Primers play a crucial role in these steps, so if the design is incorrect, it can lead to non-specific binding, hair loops and dimers. To assist others in designing a good primer, we have created a step-by-step guide for the same.

What are Primers?

Primers are short, single-stranded DNA sequences that are complementary to the target DNA region and serve as starting points for DNA synthesis. They are used in polymerase chain reaction (PCR) to amplify specific DNA sequences for a variety of applications, including genetic testing and sequencing. In PCR amplification, it is very important to have the right primers. Now, we go through the steps using an example sequence, and we design a pair of primers for it using the SnapGene application.

An Example

The above is an example homologous recombination deletion cassette, and we now design the primers for amplifying this cassette. But first, some of the major things to keep in mind while designing primers:

• Try to keep the length of the primer between 18-30 nucleotides. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.
• The melting temperature (Tm) of the primer should fall between 50-65°C. Since the Tm depends on the length of the primer, it's important to keep the primer length short. Additionally, the bases in the primer impact Tm, with G and C resulting in higher melting temperatures than A and T. If the Tm of your primer is too low, try to find a sequence with a higher GC content or slightly increase the length of the primer.
• Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding. A GC Clamp is what this is called. The stronger hydrogen bonds between the G and C bases contribute to the primer's stability. Be careful not to have too many G or C repeats, as this can result in the production of primer dimers.

First, using SnapGene, select the sequence you think would make a good primer - here we take a 25bp sequence, and its Tm is visible when you hover the mouse over the selected sequence.

Now, since this is intended to be the forward primer copy, the top strand of this sequence uses the “ctrl+c” function. (forward primer anneals to the bottom strand, so it must be complementary to the bottom strand, or in other words, the forward primer will have the same sequence as the top strand. Similarly, the reverse primer anneals to the top strand, so it's complementary to the top strand, or in other words, it has the same sequence as the bottom strand.)

After the top strand has been copied, go to the following website and select the ‘OligoAnalyzer tool’ from the 'Tools' menu.

Now, after opening the tool, paste your sequence and click on analyze. Upon clicking analysis, various properties of the primer uploaded should be displayed of which the GC content and Melt Temp (Tm) are the most important and need to be noted.

Once the Tm and GC have been noted, click on the ‘Hairpin’ option under which we see the melting temperature of the various hairpins possible for the primer.

• Make sure that the Tm of your primer must be greater than the Tm of your hairpin (try to keep a difference of at least 20 degrees Celsius). Also try to keep the ΔG below -2.

Here we see that the highest Tm possible for the hairpin is 33°C and the Tm of our primer is 57.5°C, this is a significant difference. If in case the difference is not significant enough, you can visualize the hairpin to see which nucleotide is causing the hairpin formation and try and avoid those sequence in your primer.

Now, we can also check for self dimer with the ‘self dimer option’.

After clicking on the ‘self dimer’ option we get the Delta-G scores; make sure that there is a significantly huge difference between the Max Delta G and the subsequent Delta G’s.

Now repeat the same steps for making the reverse primer: select the sequence you want as the reverse primer making sure it has almost the same Tm as the forward primer. Once the reverse primer has been checked, annotate the sequences in SnapGene as forward and reverse primer.

Select the sequence, then click on the ‘add primer’ option. Select whether it is the forward primer or the reverse primer, and then name the primer as ___.fwd or ____.rev respectively and click the ‘Add to template’ option. Then the primer will get annotated to the sequence.

Now similarly annotate the reverse primer in the sequence. Once the forward and reverse primer has been annotated in the sequence, try a virtual PCR to make sure the sequence is getting amplified correctly (Press ‘ctrl+D’ to do a virtual PCR. Or you can manually select PCR from the ‘actions’ option in SnapGene).

In the PCR window, select your forward primer in place of ‘primer 1’ and reverse primer in place of ‘primer 2’. Then click on PCR.

Now the amplified DNA file is created.

Check if the correct sequence is amplified. On clicking ‘Primers’ we get to see the properties of the primers created. Make sure that the primers have a single binding site only.

To export the primer info as a text file, click ‘Primers’ on the top tab and select- ‘Export primer data’. Also, do check the annealing temperatures of your primers based on the polymerase that you will be using.

For example if using any polymerase from NEB like Q5, check the annealing temperature using the NEBtm calculator online by copy pasting your forward and reverse primers.

After receiving the primers and doing the PCR for the first time it is recommended you perform a gradient PCR first to check at which temperature appropriate amplification occurs. Then perform the actual amplification using a bigger reaction volume.

Design of Our Gene Cassette

Our gene construct was made after careful steps of getting the sequence, promoter, and terminator, followed by codon optimization and more. Here are the detailed steps of making our gene construct. Other teams can take inspiration from the guide.

Initial Knowledge

• Each expression construct should have a promoter, coding sequence and terminator.
• CDS of JcFATA and JcFATB are available on NCBI and the truncated CvFAP sequence can be taken from ^[1].
• pTEF1 promoter and XPR2 terminator sequences can be found on iGEM parts repository.^[2][3]

These were chosen as XPR2 is very commonly used in literature and by previous iGEM teams (Calgary 2019), as a terminator for heterologous protein expression and has been widely successful. pTEF1 has also been used in literature and by iGEM teams, having many variations. We chose the smallest one to keep our construct concise.

Designing the Construct

Since we needed to clone 3 genes, 3 different plasmids being expressed would have been too stressful for the cells due to 3 selection markers. Prof. Sunish K R had suggested the existence of “Dual” vectors which allow expression of 2 genes with one plasmid when our plan was about E. coli, but we didn’t find similar expression vectors for yeast because Y. lipolytica does not have autosomally replicating plasmids.

Upon research, we went through multiple methods like Golden Gate assembly, Gibson Assembly and NEBuilder® HiFi DNA Assembly, and finally settled on NEBuilder® HiFi DNA Assembly as the most optimal, due to its high efficiency and success rate even as compared to Gibson assembly.

We needed to introduce overhangs into the constructs(approx 30 bp) according to NEBuilder® HiFi DNA Assembly, so we did that. Upon realising the limitations of free gene synthesis from Twist Bioscience, that we cannot synthesise fragments longer than 1.8 kbp at a time, we broke our CvFAP gene construct into 2, to be joined together during the NEBuilder® HiFi DNA Assembly.

For expression in Yarrowia lipolytica, we found a kit, “YLEX Yeast Expression Kit”, by Yeastern Biotech, which was invented by a leading scientist on Y. lipolytica research, Dr. Catherine Madzak. Since we have no one working with this yeast in our institute and we were in constant contact with Dr. Madzak through mail we decided to use this kit as she could help us with troubleshooting.

The way to clone our genes of interest into pYLEX is restriction enzyme based digestion ligation, and for that our gene needed to have a BamHI site on one side, according to the manual. The other side is supposed to be a blunt end ligation where the construct is supposed to start with AATG and the plasmid is supposed to be digested with PmlI restriction enzyme (recognition site CACGTG).

We wrongly interpreted this to mean that our insert needs to have both the PmlI (upstream) and BamHI (downstream) sites on its two ends, so we introduced these at the start of fragment 1 (JcFATB) and end of fragment 4 (JcFATA).

Dr. Sunish Radhakrishnan and our secondary PI Dr. Mridula Nambiar suggested to optimise the JcFATA and JcFATB genes codons to make them optimal for Y. lipolytica, so that we don’t have any rare codons according to the codon bias of Y. lipolytica. CvFAP has already been expressed in this yeast so we had the codon optimised sequence already available from ^[3].

Upon talking to our PhD mentors (Sonali, Geetanjali, Saptarshi, Kundan) they told us to ensure that we should not have multiple copies of the restriction sites that are required for the ligation as well as the linearisation of pYLEX procedure, i.e. BamHI, PmlI and NotI, in our gene construct. To ensure this, we did codon optimisation on SnapGene and IDT, to replace the codons to ensure there were no sites for these enzymes in our construct.

We read a paper on Golden Gate Assembly where they had introduced “spacer sequences” in between every component like promoter, gene, terminator in a multiple gene expression system. Our PhD mentors, Dr. Sunish and Dr. Mridula suggested that the spacer between the promoter and gene could be a Ribosome Binding site(RBS), called a Kozak sequence in eukaryotes, whereas the one between terminator and promoter were probably just to give a space between the two.

Not sure of the Kozak sequence of this Yeast, we mailed Dr. Catherine Madzak with the same query and she explained that CACA is the Kozak sequence in Y. lipolytica, and we should add that in between our promoter and gene. This also cleared our misunderstanding of adding a PmlI blunt site in our construct because we realised that if we start the construct with AATG instead of a PmlI site and the plasmid ligating upstream ends with CAC (due to digestion by PmlI) then we would get a “CACAATG” sequence, i.e. Kozak sequence followed by start codon, which is exactly what Catherine was referring to.

Apart from this, on the suggestion of our BSMS mentors, we introduced unique RE sites after each promoter, gene, terminator, in case Hi-Fi Assembly didn’t work and we were forced to resort to RE based approaches.

This was just a safeguard in case things went bad. The sites introduced here were XbaI , SpeI , BstBI, AgeI, BspDI, AflII, ApaLI.

Lab Protocols

Our project experimental section consists of a variety of protocols that we have used. We believe that lab protocols are extremely important to get good results. Our compiled protocol section has protocols for the preparation of various types of media, buffers, NEB Hi-Fi DNA Assembly, PCR amplification, Agarose Gel electrophoresis, PCR clean-up, gel extraction, colony PCR, plasmid miniprep, restriction-digestion, ligation and transformations for both DH5α E.coli cells and Yarrowia lipolytica. Yarrowia does not have a lot of standardized literature for various experiments, so one can find what we did in protocols and how we improved it in the DBTL cycle under engineering success.