Part:BBa_K5087009

ItsR

Introduction

Biology & Usage

The ItsF and ItsR primer sequences were taken from a publication of White et al. [1].

Our team utilized this primer to obtain the fragment containing the ITS2 sequence of Prymnesium parvum, measure the DNA concentration, and subsequently use it in SHERLOCK assays. This was done to establish the limit of detection for both our plate fluorescence readout test and the PrymFlow lateral flow assay. 

The ITS2 (Internal Transcribed Spacer 2) region is a non-coding segment of DNA found within the ribosomal RNA (rRNA) gene cluster. In the genome of Prymnesium parvum, the ITS2 region lies between the 5.8S and nuclear large rRNA genes [1].

The ITS regions, including ITS2, are commonly used for species identification because they tend to vary between species while being conserved within a species. This variability makes the ITS2 region an effective target for designing species-specific primers, such as those used to identify Prymnesium parvum [2].

Experimental Validation

Purpose

The purpose of this experiment was to evaluate the usefulness of ItsF and ItsR primers for PCR amplification of Prymnesium gDNA.

This was done to address previously encountered issues with the low concentration of Prymnesium gDNA. By PCR-amplifying the detected fragment, we aimed to obtain a higher concentration of the target DNA for more reliable testing and to enable precise quantification of its concentration.

Direct measurement of gDNA concentration is unreliable because our algal cultures were established from an environmental sample taken from a water body affected by Prymnesium parvum blooms, meaning they are not pure cultures and may contain DNA from various other organisms. Consequently, the gDNA isolated from these cultures likely includes DNA from these other organisms in addition to Prymnesium parvum. When DNA concentration is measured using a NanoDrop Microspectrophotometer, it quantifies all DNA present in the sample, of which Prymnesium parvum DNA might constitute only a small percentage. 

Methods

Preparation of PCR Reaction:

• We mixed the following components per reaction in a PCR tube:

• 5 μl - Q5 High Fidelity 2x Master Mix polymerase (New England Biolabs)

• 0.5 μl - 10 μM ItsF

• 0.5 μl - 10 μM ItsR

• 2 μl - nuclease-free water 

• We prepared 5 replicates for each of the primer sets: Gal (BBa_K5087000 and BBa_K5087001) primers, Kac (BBa_K5087006 and BBa_K5087007) primers and the Its PCR primers (BBa_K5087008 and BBa_K5087009).

• We assembled the reactions with 2 μl of DNA per reaction. We used the algal DNA with an approximate concentration of 35 ng/μl. For each primer set, the first reaction was a negative control with nuclease-free water added instead of the DNA.

• We gently mixed the reaction components and, if necessary, collected all liquid at the bottom of the tube by a quick spin.

Thermocycling Conditions:

• We transferred the PCR tubes to a PCR machine with a heated lid, preheated to 98°C.

• We set the thermocycler with the following parameters:

  • pre-denaturation - 98°C, 30 sec

  • denaturation - 98°C, 10 sec

  • annealing - temperature gradient (55°C, 60°C, 65°C, 70°C) 30 sec

  • elongation - 72°C, 1 min

  • final extension - 72°C, 2 min

  • number of cycles - 40

• The negative control reactions were carried out in 55°C.

Electrophoresis:

• We prepared 1 % agarose gel.

• For each sample, we mixed 5 μl of PCR product with 2 μl of a loading buffer and 5 μl of nuclease-free water obtaining a total volume of 12 μl.

• We loaded the prepared samples onto the gel, alongside 5 μl of ladder (1 kb Plus DNA Ladder, New England Biolabs)

• The electrophoresis was conducted under 90 V for 50 minutes in TBE buffer solution.

• We visualized the DNA bands under UV light. The target Prymnesium parvum product was expected to be 760 bp in length.

Results

Figure 1. PCR results in different temperature ranges.

Conclusions

A product of the approximately correct length (760 bp) has been obtained with ItsF and ItsR primers, and can be used for future experiments. The primer set works reliably in the conditions optimized by our team. The gel did not run evenly, so the bands for Kac primers appear higher up than they likely are in reality. It was also determined that the optimum annealing temperature for the Its primer set is 60°C. 

Sequence

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Biosafety

We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.

References

• [1] White, T.J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications. Academic Press.
• [2] Galluzzi, L., Bertozzini, E., Penna, A., et al. (2008). Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. *Letters in Applied Microbiology, 46*(2), 261-266. https://doi.org/10.1111/j.1472-765X.2007.02294.x