Stolen moments Vol 1: DNA + Molecular Cloning

From counting birds at Christmas to cataloguing bacteria in the human microbiome, Citizen Science is a template of scientific participation that can be co-opted by teachers to engage large numbers of students in research training.

I’m in the process of designing a brand new lab course for Semester 2, and the next series of blog entries will be my attempt to “think out loud” and steal some time away from the pressure of looming deadlines... The focus will be on inquiry-based learning in molecular biology labs classes - starting today with DNA + Molecular Cloning:

TLDR

In molecular cloning, if one step fails, everything fails. It is very tempting to have students follow cookbook recipes for each part of the process, but it is more valuable for their learning if you give students more autonomy in the experimental design. You can soften the learning curve by limiting the parameters that students can change:

Designing controls, serial dilutions, optimising concentrations for reagents…

All of this provides a scaffolded safety net for students to make inquiry-driven decisions. Lab class design is a big emphasis in the science education literature, and you can look through JMBE, BAMBED, CBE life Sciences, and FEMS Microbiology Letters to see how others have incorporated recombinant DNA work into lab teaching. Useful keywords include undergraduate research, CUREs (Course-based Undergraduate Research Experiences), or Citizen science. For each step in the molecular cloning workflow, let’s workshop how to introduce inquiry-based elements to challenge student thinking.

Inquiry Idea 1: PCR Primer Design

You can give ask students to choose from a pool of different genes and ask them to design PCR primers to amplify them. You provide the basic design principles about primer length, primers ending in G or C, overall G+C percentage, and melting temperature ranges, and students have to work it out. If you have the time and resources to order and test these student primers that’s great, but it’s not very scalable for large classes. A workaround involves three different sets of primers that you’ve already designed for every gene, and asking students to use design principles to predict which will work best.

Another exercise would involve a set of primers completely designed by an online tool, without any manual tinkering – can students beat the algorithm? Will their primers be better than the ones automatically generated by a computer? The next step is to actually run the PCR reaction and analyse the PCR products using gel electrophoresis. Students should make their own PCR mastermix and do the calculations for each reaction, design positive and negative controls for the PCR reaction, and predict the size of DNA bands to be amplified.

Inquiry Idea 2: Restriction Digest

Most labs will provide students with one consistent cloning strategy to use, just because there are too many ways this can go wrong. A slight compromise is giving students a choice of three different cloning strategies, sets of restriction enzymes, or plasmids with different tags (GFP, His-tag, GST-tags), and asking them to justify which will work best. They need to look at the multiple cloning site of the plasmids, the sequence of their gene of interest, as well as PCR primers you provided (or they designed!) to see which will work best. When setting up the actual restriction digest again it comes down to students designing appropriate controls – most likely a negative control in this case and analysing the outcomes using gel electrophoresis.

Inquiry Idea 3: DNA ligation, transformation, and screening clones

After verifying the restriction digest and cleaning up the reaction, ligating the gene of interest and plasmid together is next. Ligation reactions are notoriously finnicky, so ask students to take ownership over how many replicates to setup, the different ratios of plasmid to gene insert for each ligation reaction, and again the appropriate experimental controls. All of this then goes through DNA transformation into competent bacterial cells. You can then subculture any transformed colonies and screen them using Colony PCR. If the genes of interest are of genuine value to an ongoing research project, then there is a vested interest to validate positive clones. This makes the whole project more authentic, and one positive clone per gene can then be sequenced for them to check the accuracy base by base. I’ve previously released videos on DNA transformation, Colony PCR, and DNA sequencing, and these can be integrated as part of pre-lab exercises in a blended-learning module.

Putting it all together

If you add up all these steps - designing PCR primers, PCR reactions, restriction digests, ligations, transformations – this will take a minimum of four or five 3-hour sessions for students to do everything, maybe even more if you provide them with more responsibility in experimental design. This isn’t glamorous work but as molecular biologists we know it is foundational to our field.

That’s enough procrastination (and catharsis) for the moment… more details in the video below, and we’ll talk about protein analysis next time.

Jack.