My research group focuses on the fundamental molecular biology and biochemistry of biosynthetic assembly lines in bacteria. Our goal is to engineer bacteria to make new antibiotics. We are passionate about bringing this work into the classroom, providing students hands-on experience in cutting edge techniques in biotechnology.
Topoisomerase-based cloning is currently used in the Cal Poly undergraduate molecular biology classroom to introduce students to molecular cloning. Gibson Assembly has emerged as an industrially relevant molecular cloning technique, and we are trying to provide students with applicable experiences. Molecular cloning is a technique that can be difficult to conceptualize in a traditional undergraduate classroom.
We have designed a hands-on laboratory module in which students design and evaluate their experiment as well as contribute to authentic research projects. This enhances their understanding of the cloning process and applications of their work. While other course-based undergraduate research experiences utilizing molecular cloning techniques have been published, none include an adaptable Gibson assembly module.
We've designed all of the materials to measure the learning outcomes for this course-based undergraduate research experience. We hope this will serve as a easily implementable science education tool to improve instructor assessment and student education in college-level molecular biology laboratories. To begin, determine the DNA template source for the gene inserts, such as genomic DNA, plasmid, or synthetic DNA.
Retrieve the DNA sequences of the gene inserts and vector. Import the desired insert sequences and vector sequences into Benchling as new DNA sequence files. Then open each imported sequence to be used in the Gibson assembly reaction.
Now locate the Assembly Wizard tool at the bottom of the screen. Click Assembly Wizard, then select Create New Assembly. From the options provided, select Gibson, and click Start to begin the assembly.
In the Vector Sequence window, select the vector backbone bases starting from the 3 prime end of the gene insert to the desired endpoint. Once selected, click the Backbone tab at the bottom of the screen and choose Set Fragment. In the Insert Sequence window, select all the insert bases to be included in the assembly.
Once selected, click the Insert tab at the bottom of the screen and choose Set Fragment. If there are multiple gene inserts, click the plus button on the right side of the Assembly Wizard. In the Insert Sequence window, select all bases for the additional insert to be included in the assembly.
Once selected, click the Insert tab at the bottom of the screen and choose Set Fragment. Once all fragments are set, rename the assembly to the desired plasmid name. Click Assemble on the right side of the Assembly Wizard.
Then select the desired folder location for both the sequence folder and primer folder, and click Create to assemble the recombinant plasmid sequence. Now open the assembled plasmid and click Assembly History to view a basic plasmid map and an overview of the sequences from which it was derived. In the Assembly Parameters tab, view the names of the primers designed for the assembly.
Confirm that there are two primers for each insert and that primer names are derived from the DNA sequence file titles. Ensure that the primer melting temperatures and suggested annealing temperatures are compatible. Once the primer design for the desired plasmids is complete, click Finalize in the Assembly Wizard.
Using a gradient PCR experiment with the DNA templates, test both insert and vector-specific primer pairs for optimal annealing temperatures. Once annealing temperatures have been determined, create aliquots of primer solutions, DNA templates, and necessary reagents for PCR reactions for students. To begin, obtain primers and template DNA solutions for PCR from the instructor.
Using the reagents shown here, prepare 25-microliter PCR reactions to obtain linear fragments of desired DNA sequences. Cycle the reaction in a thermocycler. Ensure the annealing temperature is appropriate for the primers and that the extension time is suitable for the length of the desired amplicon.
Next, analyze five microliters of each reaction via agarose gel electrophoresis. While the gel is running, add one microliter of DpnI restriction enzyme to each reaction that used plasmid DNA as a template. Incubate this mixture for one hour at 37 degrees Celsius.
Then image the gel to confirm that PCR was successful and correct amplification was achieved. Purify any successful PCR reactions using a commercially available PCR purification kit. Measure the concentration of purified PCR product using a microvolume spectrophotometer for use in subsequent Gibson assembly calculations.
Pipette the Gibson assembly reaction components. Incubate the reaction at 50 degrees Celsius for 15 minutes. While the reactions incubate, thaw chemically competent Escherichia coli cells on ice for transformation.
Transform two microliters of the Gibson assembly reaction into chemically competent cells via heat shock. Pipette 100 microliters of the transformed cells onto two selection plates and spread with sterilized beads. Incubate the plates overnight at 37 degrees Celsius.
On the next day, store the plate at four degrees Celsius. Then count colonies on all plates, and calculate transformation efficiency using the given formula. Using a marker, select and label four distinct colonies on the selection plate with the student's initials and a number.
Take a new LB agar plate containing antibiotics for selection and divide it into quadrants. Use approximately half of each colony to restreak onto the respective quadrant. Add the correct antibiotic concentration to the tubes labeled with colony identity for selection.
Then inoculate a five-milliliter liquid LB culture with the other half of each of the four selected colonies. Incubate the liquid cultures in a shaking incubator at 37 degrees Celsius overnight and the agar plate in a static incubator at the same temperature. Isolate plasmid DNA from the liquid cultures using a mini prep kit.
Measure the concentration of the isolated plasmid DNA in nanograms per microliter. After designing a restriction digest PCR screen to analyze the isolated plasmids, pipette the restriction digest reactions, or PCR reactions. Then analyze the results by gel electrophoresis.
Once the Gibson assembly module is completed, ask the student participants to complete the multiple choice post questionnaire in the lab meeting. Combine all data from the pre and post questionnaire responses for analysis, and assess their statistical significance. After the project, the average content score significantly increased from 63.7%to 80.4%indicating improved student understanding of molecular biology and cloning concepts.
The average term confidence increased significantly from 3.52 to 3.87, with a large effect size demonstrating improved student understanding of molecular biology terms. The average student's confidence in performing lab techniques increased significantly from 3.82 to 4.33, showing enhanced comfort with molecular biology techniques. The number of responses indicating familiarity with the term Gibson assembly significantly increased from pre to post questionnaire, with a notable rise in those who could explain the term.
Confidence in performing the Gibson assembly technique improved with most responses, shifting from neutral or disagreeing before the session to agreeing or strongly agreeing after. Student confidence in general biology topics showed no statistically significant changes. In contrast, confidence in specialized Gibson assembly questions increased significantly.
