May 15 – 19, 2023

Prior to Workshop Meetings

We will send each of you a link to a Google Slides document. Each participant should prepare one slide to introduce themselves to the group by addressing prompts in the slide and posting recent photograph.  A beanbeetles.org website link also will be provided that will contain direct links to every protocol and video mentioned in this agenda.

Read the protocols on surface sterilizing beetles and preparing media for microbiome culturing [instructor’s notes][student handout].  Prepare the following:

• 15mL of 10% beach in a 15ml test tube or conical tube
• 15ml of 70% ethanol,
• 30ml of sterile water (tap or deionized) in two 15ml tubes
• Rack for four tubes to be held in a row
• Prepare 100mL of media for 6 or more nutrient agar plates in 100mm Petri dishes (and selective media if available or order some prepoured plates) and hold in a refrigerator until the first day of the workshop (Monday). If you are working alone, need only 3 plates of nutrient agar.

Tuesday 16 May:  Plates and surface sterilization tubes will be used on Tuesday of the workshop.  You will need:

• Micropipetters P1000 [200-1000uL], P200 [20-200uL] and P20 [2-20uL] and sterile tips for each
• 8 sterile microfuge tubes (1.5ml)
• 100ml of 0.9% sterile saline (0.9g NaCl in 100ml water, then autoclave), dispensed in sterile 1.5ml microfuge tubes containing 1.0ml each per person.
• cell spreader(s) to prepare culture plates (glass or metal spreader must be alcohol dipped and flame sterilized prior to each use, or use disposable plastic spreaders, minimum 3 per person).
• Vortex shaker for mixing microfuge tube contents
• autoclave bag for waste 

We will send you a live bean beetle culture, soft forceps to handle beetles, and sterile microfuge tube pestles. 

Wednesday 17 May:  Read the protocols for PCR [instructor’s notes][student handout] and gel electrophoresis of PCR products [instructor’s notes].  On Wednesday, you will need:

• PCR thermocycler
• micropipetter P20 and sterile tips
• Vortex shaker
• Molecular grade sterile water (240uL needed for a total of 4 PCR reactions)
• PCR tubes to conduct PCR (one strip of 8)

We will send a tube of PCR mastermix with primers and loading dye for 16S rRNA gene amplification. 

You will evaluate the PCR products using gel electrophoresis and will need:

• 1% agarose gel in TAE buffer (50ml for one gel with 8 wells, 0.5g agarose in 50ml 1x TAE))
• TAE buffer 1x, for making 1% agarose gel and for running buffer, approximately 300mL
• DNA stain (GelRed is the safest), 5uL
• DNA ladder (100 – 1500bp), 7uL
• Micropipetter P20 and tips
• UV transillumintor

Workshop

Monday

1:10-1:20pm EST          Introductions

1:20-1:40pm                 Discussion on Workshop Expectations and Implementation Process [presentation]

1:40-2:10pm                  Discussion on CUREs in laboratory classes

Each workshop will start with a session on inquiry-based learning, defining CUREs in laboratory classes, and the value of CUREs. We will discuss the different approaches to inquiry-based learning, ranging from guided-inquiry to open-ended inquiry to CUREs. In addition, we will discuss definitions of CUREs based on participants’ responses compared to other prior definitions, and how research in laboratory courses affects student outcomes.

2:10-2:15pm                 Standup and Stretch

2:15-2:45pm Importance of Microbiomes and Insect Microbiomes [presentation]

2:45-2:55pm                 Break

2:55-3:25pm                  Bean Beetles as a model system [presentation]

In this session, we will introduce participants to bean beetles as a model system for teaching and research. Participants also will be introduced to our website resources (www.beanbeetles.org). These resources include a handbook on the use of bean beetles, links to websites of researchers who study this species, extensive bibliographies of research articles on the genus Callosobruchus and other bruchid beetles in different sub-disciplines of biology, more than thirty-five class-tested inquiry-based laboratory activities using Bean Beetles complete with student handouts, instructor’s notes, and sample student data, and links to resources on inquiry-based learning.  Participants will develop expertise in handling Bean Beetles, sexing adults, identifying eggs on beans, and culturing beetles.

Homework: 

• Read two of the assigned research articles about microbiomes and pose a question (to be discussed on Tuesday) that you and your students might address on microbiome communites in bean beetles.
• Read the protocols [instructor’s notes][student handout] and handling beetles, surface sterilizing beetles, and extracting microbiome samples for colony culture plating. 

Tuesday

1:10pm-2:00pm EST      Bean Beetle microbiome protocols 1

During this session, we will begin by simulating the process of eliciting student-generated research questions based on background reading, followed by student-generated experimental design.  Then, workshop participants will discuss the bean beetle microbiome CURE protocol.

2:00-2:10pm                  Break

2:10-2:30pm                  Breakout rooms for Small Group Discussions about selecting research questions

2:30-2:35pm                 Standup and Stretch

2:35-3:25pm                  Bean Beetle microbiome protocols 2 [presentation]

            We will continue discussion on the three experimental components of the bean beetle microbiome CURE.  One of those components, colony phenotype analysis, involves identifying bacterial colony phenotypes and tabulating the number of colonies of a given phenotype on culture plates.  We will practice this tabulation with images of colonies on culture plates, and tabulate your findings. [Spreading with glass beads][Glass beads]

Homework:

• Prepare a microbiome extract following the direction in the Microbiome Culturing Protocol [instructor’s notes][student handout] and plate extract on nutrient agar plates (and PEA and EMB plates if available) to culture overnight. 
• Read the protocol on conducting a community ecology analysis on colony phenotype data [student handout] and perform this analysis on the sample class dataset.  You can create a larger dataset for analysis by searching on colony phenotype data based on sample metadata and colony phenotype.
• Read protocols on PCR [instructor’s notes][student handout], BLASTn [student handout] and analysis of Sanger sequence data [student handout] and view video demonstrations on these protocols [What is PCR? Polymerase Chain Reaction | miniPCR bio™][What is Gel Electrophoresis? | miniPCR bio™][Introduction to Sanger Sequencing][Colony-based PCR][Electrophoresis of PCR products][Conducting BLASTn Searches on Sanger Sequences].

Wednesday

1:10-2:00pm EST                      Microbial community ecology analyses 1 [presentation]

Colony phenotype, colony-based 16s rRNA sequence results, and high throughput sequence results can be used to describe microbial communities. In this session, we will guide participants in a discussion on the community-level ecological analyses that can be calculated (such as species richness, species diversity and other methods) and how they may be interpreted.  We will address questions and problems you may have had with the analysis that you should have conducted as homework last night.  We also will discuss how to interpret the results and the limitations of this analysis. [Excel completed analysis][Google sheets completed analysis]

2:00-2:10pm                              Break

2:10-2:40pm                              Bean Beetle microbiome protocols 3 [presentation]

Participants will examine the bacteria cultured the previous day (homework).  We will discuss the protocols for picking a bacterial colony for PCR amplification of the 16S rRNA gene.  The PCR product of picked colonies can be Sanger sequenced, and the sequence identified to genus using NCBI BLASTn.  We will discuss the process of colony picking and preparing a PCR run.  We will show images of electrophoresis gels of the PCR products and review how to interpet those results to confirm successful PCR amplification.  We will then show how Sanger sequence data may be used to identify each picked colony to the level of genus using NCBI BLASTn and assemble a colony sequence dataset for community ecology analysis. [sample .ab1 file][TEAL][sample fasta data][BLAST]

2:40-2:45                                  Break

2:45-3:15                                  Tech Preparation for NextGeneration Sequence Analysis

Each participant will create a CyVerse Account and communicate their user name to us.  That will permit us to share a Demo Data file with each of you for use in DNA Subway. [Cyverse Tutorial]

Homework:

• Pick a bacterial colony from your culture plate to prepare and run PCR [instructor’s notes][student handout].  Then, perform electrophoresis on the PCR products [instructor’s notes]. 
• Read protocol on whole microbiome DNA extraction [instructor’s notes][student handout] and view the video on this procedure [DNA extraction]. 
• Read the protocol on the community ecology analysis of colony-based sequences [student handout].
• Perform the community ecology analysis of the colony-based sequences (Sanger sequences) using the sample class dataset. You can create a larger dataset for analysis by searching on colony-based Sanger sequence data based on sample metadata, colony phenotype, and bacterial taxonomy.

Thursday

1:10-2:00pm EST                      Microbial community ecology analyses 2

In this session, we will discuss the community ecology analyses you performed on Wednesday may be used to evaluate the colony-based sequence data the 16s rRNA gene (from Sanger sequencing).  We will address questions and problems you may have had with the analysis of the sample dataset you should have conducted as homework last night.  Both small datasets and large datasets may be evaluated.

2:00-2:10pm                              Break

2:10-3:00pm                              Microbial community ecology analyses 3 [presentation]

This session of the workshop will focus on the kinds of data that students will receive from whole community sequencing (NextGen) of Bean Beetle microbiome preparations. We will guide participants through processing the sequence data by providing sample data. Participants will begin work through a tutorial designed for teaching undergraduate students how to carry out analysis of microbial community sequencing data using the DNA Subway computer application. [DNA Subway tutorial][DNA Subway Video tutorial]. 

Homework:

• Complete the DNA Subway tutorial and download a csv Level-5 Taxonomy File from DNA Subway. 
• Read the RShiny BeanBeetleMicrobiome Tutorial and perform that analysis using the sample dataset. If you were unable to complete the DNA Subway analysis, you can download the files that you will need here. [level-5][level-5-transposed][metadata]  
• Discuss your implementation plans with your institutional partner and list the potential barriers you may have in your implementation. Each team should prepare one slide to present your implementation plan (use Google Slide on Implementation).

Friday

1:10-2:00pm EST           Microbial community ecology analyses 4

In this session we will address questions and problems you may have had with the processing of next-gen sequence data in DNA Subway and the analysis of the sample dataset you should have conducted with the RShiny app as homework last night.  We also will discuss the interpretation of the output of the RShiny BeanBeetleMicrobiome app. 

2:00-2:10pm                  Break

2:10-2:40pm                  Implementing the Bean Beetle microbiome CURE and overcoming barriers

This session will dedicate time for each faculty team to present implementation plans. We will also guide faculty in data management and how they will incorporate their data into a network-wide database.  In addition, we will facilitate a discussion with the workshop participants on overcoming barriers to implementing CUREs in the participants’ laboratory courses.  This will permit feedback from the entire group and allow faculty to see similarities among ideas.

2:40-3:10pm                  Using student assessment to guide and foster pedagogy change [presentation]

In this session, we will discuss the opportunities that participants have to document the benefits of CURE pedagogy with their specific student population. We will discuss how participants may use easily collected student outcome data to foster broader curriculum change in their academic department and more broadly in other STEM disciplines within their home institution.

3:10-3:20pm                  Break

3:20 – 3:50pm               Next steps and workshop assessment

Zoom meeting link will remain open each day until 5:00pm EDT for participant initiated breakout meetings and collaborations following the scheduled workshop sessions.