iGEM Sheffield 2023: Transformation of E. coli with secondary school students
During iGEM Sheffield's previous outreach efforts to local secondary schools, it became apparent that whilst biotechnological approaches and theory are present in the syllabus for A-level Biology, there is significant lack of any hands-on practical experience with everyday molecular biology. Since aptitude in a wide range of molecular techniques is vital for synthetic biologists to have the fidelity to manipulate biomolecules, iGEM Sheffield 2023 were keen to provide A-level students (aged 16-17) with the opportunity to perform a transformation of a red fluorescent protein (RFP) into Escherichia coli. To prepare for this activity, pET28a containing the mCherry (an RFP) open reading frame were created, alongside competent DH5a and a myriad of LB agar plates.
Initially, we provided students with 1 ml aliquots potassium ferrocyanate: an innocuous bright yellow solution, with which they could practise the use of automatic pipettes by transferring volumes of a bright liquid to a balance and measure their accuracy by measuring the weight of their droplets.
Students were provided with: an LB+Kanamycin plate for the transformant selection and an LB plate with no antibiotic, with which they could visualise the importance of aseptic technique by laying a fingerprint on it before and after hand sanitisation, and another plate for the “painting” of pre-transformed fluorescent bacteria. The general protocol used for transformations was as follows:
- Add 1 ng of purified RFP DNA to the competent DH5α cells on ice. Leave on ice for 20 mins.
- Heat shock at 42 C for 45 seconds
- Return to ice for 5 mins.
- Add 1 ml of LB and allow to recover at 37 C, shaking, for 1hr.
- Concentrate the cells by centrifuging
The protocol was demonstrated to the students, step by step, with a 'follow along' presentation that was transmitted to the AV system in the teaching labs, with each step illustrated by an easy to interpret diagram and an in person explanation both at the beginning of each step, and during each step via patrolling team members who could provide support. Before experimental work began, and during relevant steps, students were reminded of the essential safety considerations necessary when dealing with infectious waste, fire and hazardous substances - this was also covered in a risk assessment generated in conjunction with the Perak Teaching Laboratories.
In the interim of E. coli recovery (1 hour at 37 C), we filled the time with a presentation that detailed the importance of E. coli and other model organisms such as Synechocystis, Saccharomyces cerevisiae and HeLa cells to molecular biology research - and emphasised the critical role that genetic manipulation of E. coli plays in virtually every area of molecular biology. To reiterate this further, a few of the team members exemplified this importance by giving a brief overview of their research with attention drawn to where the use of genetically engineered E. coli was important, with the overall goal being the contextualisation of the practical work they were performing.
As the practical steps were finishing, a number of educational tasks were provided for the students. Wandering team members asked questions about the theory behind the practical work - what enzymes would be required to assemble the plasmid, replicate the plasmid etc - that was relevant to their syllabus and thus their impending exams. Additionally, questions that demanded critical thinking beyond the scope of A-level was provided, such as the question of self-ligating plasmids and how to reduce them - to which a surprising number of students correctly identified the need for using 2 different restriction enzymes when isolating an insert. To finish the day, students were provided with a set of A-level style examination questions that were loosely tied to the practical work performed.
To summarise, the outreach day was a great success - it gave students who otherwise wouldn't have the opportunity to perform molecular biology techniques the chance to do so in a real, working laboratory setting. It complemented their learning well, and the team are already in talks to make the class an event that is held by iGEM Sheffield annually.
Link to: Presentation used for outreach day