Inquiry-based learning is a form of active learning where students obtain the skills they need 97
to problem solve and make unique discoveries about the natural world(1-3). In contrast to teacher-98 centered instruction, where facts are disseminated to students, inquiry-based learning encourages 99 students to foster their own independent learning with the assistance of the instructor(1-3). In 100 addition, inquiry-based learning puts emphasis on students developing science process skills, such 101 as making observations, developing hypotheses, and formulating conclusions(1-3). 102
Course-based Undergraduate Research Experiences (CUREs) are a form of inquiry-based 103
learning that provide students with a genuine research experience. Students enrolled in CUREs 104
develop or are given a research question with an unknown outcome, use the scientific method to 105 address the question, collect and analyze data, and communicate their results (3) (4) (5) . Students that 106 participate in a CURE learn the necessary skills and techniques they need to carry out the tasks 107 required(6-8), and at the same time gain confidence in their ability to engage in the scientific 108 process(9-11). Assessment of student learning gains reveal that CUREs improve student ability to 109 think critically, interpret data, communicate results, and collaborate as a team, compared to 110 traditional lab courses (12) (13) (14) (15) (16) (17) . A critical aspect of independent research is obtaining the foundational 111 skills and introductory training needed for understanding a specific system and/or research topic of 112
interest. 113
Here we describe a simple laboratory module employed in the first half of our upper division 114 undergraduate CURE on developmental genetics, which is used to prepare students for independent 115
inquiry-based group research projects that occur in the second half of the course. In this module, 116 students are introduced to the research organism, Caenhorhabditis elegans (C. elegans), to explore 117 the concepts of RNA interference (RNAi) and codon optimization. C. elegans offers many advantages 118 that make it an ideal research organism, such as a fast life cycle, large brood sizes, and easy access to 119 genetic manipulation by forward and/or reverse genetic approaches (18) (19) (20) . Additionally, they are 120 transparent, which allows for visualization of all tissue types, and the real-time visualization of 121 fluorescent-tagged reporter proteins expressed in various tissues of interest (21, 22) . Using the 122 protocols outlined in this paper, students will cultivate and utilize C. elegans to conduct an RNAi 123 experiment where they will visualize first-hand how RNAi depletes a gene of interest and how codon 124 optimization significantly impacts gene expression. 125
The importance of this module is that it not only teaches concepts in molecular genetics and 126
introduces students to a model system, it also promotes higher-order thinking. With the instruction 127 provided here, students will gain experience and expertise in working with C. elegans, basic 128 microscopy, data analysis, and science communication. Students will have an intuition for probing a 129 research question, developing hypotheses, planning experiments, thinking about possible results, 130
and formulating conclusions. Therefore, modules like the one presented here have a positive impact 131 on student development and at the same time provide the prerequisites needed for success in STEM-132 related fields and beyond. 133
Overall, this module can be used at various educational levels to enhance students' interest 134
in science and provide the groundwork needed for independent scientific research. The module 135
incorporates active learning styles of instruction that significantly enhance student performance and 136 encourage engagement. Finally, this module can also be used as a "stepping-stone" or "bootcamp" 137 exercise to provide students with a set of skills and tools for the inquiry-based module of a CURE 138 using C. elegans as a model organism.
Intended Audience
This laboratory module was employed in the first half of upper-level undergraduate 142 developmental genetics at Stony Brook University. Most students enrolled in the course were Juniors 143 or Seniors; however, the module can be implemented at various educational levels (i.e. Freshman or 144
Sophomores or first-year graduate students as a "bootcamp" exercise). It can also be adapted for use 145 in a high school setting where students can be introduced to a model system commonly studied in 146 the life sciences, learn concepts that pertain to gene expression and regulation, and reinforce their 147 prior knowledge of the scientific method. 148 149
The module requires four lab sessions of approximately 3 hours each. We found this was 151 ample time for students to become accustomed to working with C. elegans and proficient in the 152 necessary skills needed to complete the module. However, the module timing can be adjusted as 153 needed to any desired length of time. and animal development. It is highly encouraged that students have familiarity with basic laboratory 158 procedures, such as micropipetting and sterile techniques. Prior to the module, all necessary 159 materials and information needed to complete the assignments will be provided, and students will 160
receive an introduction to C. elegans, RNAi, and basic microscopy. activities, polling questions, and independent-learning activities. To complete the life history stages 175 assignment and GFP RNAi experiment discussed below, students were arranged into groups, which 176
fostered peer-to-peer communication, teamwork, and promoted student engagement. Moreover, 177
prior to the GFP RNAi experiment, students were assigned a GFP RNAi worksheet (see below) which 178
prompted independent thinking about the experiment. By completing the GFP RNAi worksheet, 179 students had an opportunity to complete a task on their own without supervision and troubleshoot 180 through any obstacles they may have encountered.
Student assessments were conducted at multiple levels throughout the module. During the short 184 introductory lectures given, students were asked a series of polling questions incorporated into the 185 lecture (Supplement 1) and were assessed based on their answers. Students were evaluated on their 186 ability to perform lab tasks and follow directions given by teaching assistants and instructors. 187
Additionally, students were graded based on the quality of their lab assignment, which included data 188 analysis, figure generation and figure legend writing (Supplement 2).
Inclusive Teaching 191 We have designed this module to be all-inclusive by differentiating content and lesson material to 192 reach various types of learners. The hands-on activities of this module capture the attention and 193 engagement of kinesthetic and tactile learners. Our short lectures that contain images, provide 194 written instruction, and facilitate discussion amongst the class are accommodating to both visual and 195 auditory learners. In addition, we encourage splitting the class into groups to include both men and 196
women, as well as students of different ethnic backgrounds to foster an inclusive instructional 197 environment. Thus, the module ensures equity by reaching all types of learners and encourages 198 diversity through the formation of disparate groups. Overview of the module 204 In the module discussed in detail below, various concepts that are central to the 205 understanding of gene expression and gene regulation will be explored. Using C. elegans as a model 206
organism, students will understand how codon optimization of a nucleotide sequence significantly 207
impacts the effectiveness of RNAi-mediated gene depletion. 208
Specifically, students will work with two GFP-expressing C. elegans strains, where one strain 209 expresses a non-codon optimized (NCO) GFP tag (GFPNCO), while the other strain expresses a codon 210 optimized (CO) GFP tag (GFPCO). The GFPNCO and GFPCO tags are each fused to the histone protein, his-211 58 (H2B), that is driven by a ubiquitous promoter, eft-3, which promotes expression in all cells. 212
Students will treat each strain with an empty vector (control) RNAi bacterial clone or an RNAi 213 bacterial clone that produces dsRNA specific to only the non-codon optimized GFP variant (GFPNCO) 214
(Review Timmons and Fire, 1998 for a detailed description on how RNAi works in C. elegans). 215
Through fluorescence microscopy, students will visually see differences in GFP expression in each 216 strain due to codon optimization, and they will observe that significant depletion occurs only in the 217 strain expressing eft-3>H2B::GFPNCO. From their understanding of RNAi and codon optimization, we 218 anticipate that students will be able to accurately predict these results and explain why these 219 outcomes occur. 220
Prior to the module, we present students with a series of lectures that include an introduction 221
to C. elegans and discussion about gene regulation (Supplement 1) (Review Corsi et al., 2015 for a 222
comprehensive overview of C. elegans). We discuss the topic of RNAi, which is a biological process 223 that in the presence of exogenous double stranded RNA (dsRNA) results in post-transcriptional gene 224
silencing (19, (27) (28) (29) . One method used to administer C. elegans with dsRNA is to feed them with E. 225
coli expressing a vector capable of producing dsRNA, that is complementary to a target gene of 226 interest(30, 31). C. elegans are unique in that they have a systemic RNAi response, meaning that 227 dsRNA spreads throughout all tissues, with the exception of most neurons(32, 33). Thus, loss-of-228
function phenotypes for genes of interest can be assessed in almost any tissue of interest using RNAi. 229
We also engage our students by having a discussion on codon optimization, or the 230 modification of a sequence of DNA such that the frequency of codons used by a particular organism 231 for a specific amino acid is taken into account(34-36). Codon optimization significantly enhances the 232 expression level of a particular protein due to the correlation between codon usage and tRNA 233 abundance, and mRNA stability(37-40). Thus, the expression levels of codon optimized genes will be 234 more robust than those of non-codon optimized.
In all, we anticipate this module will fulfill several goals, which include student proficiency in 236 using the scientific method and development of critical thinking skills. After completing this module, 237 students will be able to conduct controlled experiments using a model organism. In addition, they 238
will be able to explain what RNAi is and how it can be used to assess loss-of-function phenotypes for 239 any gene of interest. Lastly, students will be able to state the importance of codon optimization as it 240
pertains to gene expression. and 4). An additional lab session was devoted to an introduction to compound light microscopy and 262 a tutorial on microscopy. After the conclusion of these introductory sessions, students demonstrate 263 their ability to work with C. elegans and operate a compound light microscope available in class, by 264
imaging the life history stages (different larval stages) of the worms. Additionally, students compile 265 a figure containing images of the life stages along with a descriptive figure legend (Supplement 2 and 266
3). We find this exercise extremely valuable for students to master important worm husbandry 267 techniques (i.e. worm picking), identify larval stages, and become familiar with microscopy 268
techniques, all of which will be necessary to successfully perform the GFP RNAi experiment. To prepare the students for the experiment, we presented a short lecture on RNAi and codon 273 bias (Supplement 1) and devised a "GFP RNAi worksheet" (Supplement 5). The goal of this worksheet 274 is to drive students to formulate hypotheses as to whether the GFPNCO RNAi clone will efficiently 275 knock down GFP intensity levels in the strain expressing H2B::GFPCO or H2B::GFPNCO. In this 276
worksheet, the students are provided with the nucleotide and amino acid sequences for the codon 277
and non-codon optimized H2B::GFP tags, as well as the short-interfering nucleotide sequence from 278 the GFPNCO RNAi clone (Supplement 5). Using the sequences provided, students will make a pairwise 279 sequence alignment, using EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/). 280
They will then compare the percent similarities between the different sequences and determine 281
whether the short-interfering nucleotide sequence for GFPNCO RNAi is most similar to H2B::GFPCO or 282 H2B::GFPNCO. Through this process, students will see that the siRNA sequence (in DNA form) encoded 283
by the GFPNCO RNAi clone is 100% identical to the GFPNCO sequence and not the GFPCO sequence. 284
Students will also appreciate that the control RNAi clone is called "empty vector" because it does not 285 produce a dsRNA product. 286
To conduct the RNAi experiment, the students should grow up both the eft-3>H2B:: GFPCO When NGM plates are full of gravid adults (>100 adults on each plate or food source is near 296 depletion), students should treat each strain with alkaline hypochlorite solution (Figure 1 Step 297 1)(Supplement 3 and Supplement 4 Section V) to create synchronized L1s. Approximately 50-100 L1 298 animals should be pipetted onto control and GFPNCO-specific RNAi plates (Figure 1 Step 2). 299
Individual RNAi plates should have no more than ~50-100 worms to prevent overcrowding and 300
depletion of the E. coli food source (Figure 1 Step 2). The L1's are then cultured on the RNAi plates 301
at the desired temperature until the L3 or L4 stage is reached (Figure 1 Step 3). Once the desired 302 stage is reached, students mount the animals on 5% agarose slides containing a droplet of M9 buffer 303
and sodium azide to anesthetize the animals (Figure 1 Step 3). We recommend that students pick 304 ~10 animals for imaging at a time. We also encourage instructors to ensure students handle sodium 305 azide with care as it is toxic(44)(Supplement 3 and Supplement 4 Section VI). 306 307
Students quantified H2B::GFP fluorescence depletion using two wide-field epifluorescence 309 microscopes, the Accu-Scope or Leica DMLB fluorescence microscopes (Figure 1 Step 4 imaged for each RNAi treatment (control and GFPNCO). From the data acquired by the students, 314 several qualitative observations were made (Figure 2 A and B) . First, the overall fluorescence 315 intensity of the GFPCO strain was visually much brighter than the GFPNCO strain. Second, treating the 316 GFPNCO strain with GFPNCO RNAi strongly reduced the fluorescence intensity of GFP, whereas treating 317 the GFPCO strain with GFPNCO RNAi did not (Figure 2 A and B, eft-3>H2B::GFP column) . Third, in the 318 GFPNCO strain treated with GFPNCO RNAi, although the fluorescence intensity of GFP was strongly 319 reduced, some nuclei still showed high levels of GFP, these correspond to the cells that are insensitive 320
to RNAi, most notably neurons (Figure 2A , eft-3>H2B::GFPNCO; GFPNCO RNAi) 321
To analyze the data quantitatively, we instructed students to quantify whole-body GFP 322 fluorescence intensity for 10 animals from each strain grown on control and GFPNCO RNAi, using 323
Fiji/ImageJ2(26). Briefly, the entire body of each worm was outlined and the mean fluorescence 324 intensity (MFI) was then measured for both GFP and an area of background. The background MFI 325 measurement was then subtracted from the GFP MFI measurement to reduce background noise and 326 obtain a mean gray value (MGV). Mean gray values were normalized by dividing the MFI in RNAi-327 treated animals by the average MFI in control-treated animals (Supplement 6, Supplement 4 Section 328 VII, Tutorial Videos 1-4, Supplement 7). The mean gray values obtained from each imaging system 329 (microscope) are plotted next to their respective micrographs (Figure 2 A' and B') .
By plotting the normalized MGV, students were able to clearly see that treating the GFPNCO 331 strain with GFPNCO RNAi significantly reduced the expression of GFP compared to control-treated 332 animals (Figure 2A, 2A' , and 2B, 2B', NCO strain; control RNAi vs. GFPNCO RNAi). Moreover, the 333 students noted that treatment with GFPNCO RNAi had no effect on GFP expression levels in the codon-334 optimized strain (Figure 2A, 2A', creating a publication quality figure similar to that described for the life history stages, which also 339 included a table of their raw data and description of their results (Supplement 2 and 6). From these 340 results, and the results obtained from the GFP RNAi worksheet, it becomes evident that RNAi 341 specificity is largely dependent on the sequence homology/similarity between the target gene 342
sequence and the sequence of the siRNA produced by the RNAi clone itself.
Extended Results (Optional)
Compared to wide-field epifluorescence microscopy, high resolution fluorescence 346 microscopy greatly improves the detail and resolving power of fluorescence micrographs, such that 347
unwanted out-of-focus light is significantly reduced, and the detail of cellular objects is greatly 348 enhanced(45). Thus, to show students high quality images of nuclear DNA labeled with H2B::GFP, we 349 acquired spinning-disk confocal images for both the eft-3>H2B::GFPCO and eft-3>H2B::GFPNCO strains 350 ( Figure 2C, 2C', 3, and 4) . Importantly, these spinning-disk confocal images served to better 351 illustrate some of the key concepts discussed in the lab module, such as codon optimization and 352 lineage specific differences in RNAi susceptibility.
From the confocal fluorescence micrographs, it becomes more apparent that treatment with 354 GFPNCO RNAi significantly reduces GFP fluorescence intensity in the GFPNCO strain, but not in the GFPCO 355 strain ( Figure 2C , 2C'; CO strain vs. NCO strain; GFPNCO RNAi vs. control RNAi). To highlight the 356 differences in expression levels between codon optimized and non-codon optimized H2B::GFP fusion 357 proteins, we took spinning disk confocal images of the C. elegans germline. In general, codon 358 optimized transgenes are more robustly expressed in the germline than non-codon optimized 359
transgenes (46, 47) . In line with this, H2B::GFP fluorescence expression was more robust in germ cells 360
when GFP is codon-optimized as opposed to when it is non-codon optimized (Figure 3 ; CO strain 361 vs. NCO strain).
In C. elegans, certain cell lineages show different sensitivities to exogenous dsRNA. For 363 example, neurons are less sensitive to RNAi compared to other somatic tissues (32, 48) . This is 364 because neurons lack the dsRNA-gated channel, sid-1, which promotes the uptake of dsRNA into cells 365 that express it(49). To emphasize this concept to students, we acquired spinning-disk confocal 366
images of nuclei from various cell lineages commonly studied in C. elegans (Figure 4A) with GFPNCO RNAi significantly reduced GFP fluorescence intensity levels in the GFPNCO strain, but not 370 in the GFPCO strain ( Figure 4B-E) . However, with respect to the GFPNCO strain treated with GFPNCO 371
RNAi, the percent decrease in GFP intensity levels in the pharyngeal neurons was much less than the 372 decrease found in the other cell types examined ( Figure 4B compared to Figures 4C-E) . Thus, these 373 observations can be used in the classroom to clearly illustrate to students that certain cell types are 374 more or less sensitive to exogenous dsRNA, and this sensitivity is in part dictated by the surface 375 proteins these cells express (49). 376 377
The laboratory module presented here teaches a variety of common techniques employed by 379 C. elegans researchers and exposes students to various concepts in molecular genetics and 380 microscopy. During this module, students will become proficient at working with a widely used 381 research organism, be able to conduct controlled experiments, analyze data, produce publication 382 quality images, and have a basic understanding of microscopy. In addition, students will have a solid 383 foundation as to how RNAi works, how it can be used to study gene function, and the importance of 384 codon optimization on proper gene expression 385
This module clearly illustrates that certain cell types are less or more prone to the effects of 386 dsRNA treatment, and that codon optimization results in improved gene expression in tissues (i.e. 387
the germline). The advantage of using a strain that drives ubiquitous expression of H2B::GFP is that 388 it is extremely bright and nuclear localized, and therefore easily visible on widefield epifluorescence 389 microscopes, which are commonly available in most laboratory classrooms. For classrooms that have 390 access to a high resolution microscope, such as a spinning-disk confocal, laser-scanning confocal 391 microscope, or structured illumination microscopes, this module can be easily adapted for use on 392 those types of microscopes as shown in Figures 2C, 3 , and 4. The additional benefit of the strains used 393
in this module is that students can immediately see differences in depletion between H2B::GFPCO and 394
H2B::GFPNCO upon GFPNCO RNAi treatment. Such is a bonus when attempting to keep students 395 intrigued and engaged.
Upon completing this module, students will acquire the basic foundational skills needed for 397 independent inquiry-based research projects involving C. elegans. Some examples of inquiry-based 398 research projects that can follow this module include a reverse genetics screen to identify genes 399 important for specific processes of interest, such as longevity. In this example, with the assistance of 400 their instructor, students can design a simple research question, such as "Do fat metabolism genes 401 play a role in regulating lifespan?". Students can search the literature for fat metabolism genes of 402
interest, use either the Ahringer or Vidal RNAi libraries (Source Bioscience) to isolate clones specific 403
for those genes, and determine if their depletion reduces or enhances longevity. Results can then be 404
documented, written up in research paper format, and presented to the class. The independent 405
inquiry-based research projects that follow this module are limitless and can focus on key areas of 406 research implicated in a wide range of cellular processes, such as cell cycle regulation, cellular 407
invasion, stress-resistance pathways, vesicle trafficking, and much more. 408
Whereas most lecture and laboratory-based classrooms use expository styles of instruction, 409
classrooms that utilize active learning styles of instruction, such as inquiry-based learning strategies, 410
significantly enhance student performance and learning outcomes (15, 50, 51) . Several active 411
learning strategies that can be implemented throughout this module include variations of the Jigsaw 412 technique(52) and Think-Pair-Share(53). With respect to the Jigsaw technique, students can be 413 divided into several teams, with each team focusing on identifying a specific life history stage as 414 described above. When those teams complete the assignment, the class is divided once again into 415 new groups. Each new group consists of one member from the original teams, with each member 416 being responsible for teaching the group how to identify their originally assigned life history stage. 417
We performed a variation of the Jigsaw technique and found the students to be engaged and excited 418
with the task provided. To further promote student thinking and awareness, an instructor can also 419 take advantage of Think-Pair-Share. Here, an instructor can pose a question, allow the students to 420 think independently about the question, then form pairs or groups to discuss their ideas collectively, 421
and share with the class. This strategy can be easily administered for the GFP RNAi worksheet. Here 422 students can be given the worksheet as a homework or in-class assignment to think independently 423 about which strain the GFPNCO RNAi clone will be more efficient against. Later in the lab session or 424
during the next lab session, the students can form pairs or groups, discuss their opinions with one 425
another, and then present them to the class. In all, there are various active learning strategies that 426
can be implemented in this module, which fosters peer-to-peer communication, promotes student 427 engagement, and stimulates higher-order thinking.
This module can be further adapted for remote teaching and online learning. Instructors can 429
teach image analysis alongside with their lectures online and provide students with previously 430 acquired raw data sets from epifluorescence and/or confocal microscopes. Lessons can be held 431 synchronously by utilizing the share screen option in video conferencing apps, such as Zoom or 432
Google Meet, or asynchronously by recording lectures and lessons ahead of time, and uploading them 433
onto Blackboard, Microsoft Cloud, or Google Drive along with our image analysis video tutorials 434
(Supplemental Tutorial Videos 1-5). Based on the knowledge gained from the lectures, compiled raw 435 data, and the GFP RNAi worksheet, students will be able to formulate their hypothesis and test it by 436
analyzing the supplied data. We adapted this distance learning technique for the second half of our 437 course during the SARS-CoV2 pandemic in the Spring of 2020 and received positive feedback from 438 our students about the adaptability of the course. 439
An additional advantage of this module is that it not only applies to the undergraduate setting, 440
it can be adapted to a variety of educational levels, such as the high school or graduate level. At the 441 high school level, this module can enhance critical thinking, promote independence at an early stage 442 of a student's career, and instill awareness by introducing the field of genetics and organismal 443 biology. Moreover, it inherently promotes student engagement, by allowing students to work with 444 an organism that most are unaware exists, work in groups to share ideas, and visualize cellular 445 processes live. Additionally, we provide simplified protocols and instructions to make it easy for 446
instructors who have little experience with C. elegans to facilitate this module in their classroom. At 447 the graduate level, this module can be particularly useful for graduate student rotations and can serve 448
as an introductory "boot camp" or "stepping-stone" to introduce the C. elegans and experimental 449
techniques used in C. elegans research. Here, entry-level graduate students who have not previously 450 worked with C. elegans will have the opportunity to do so, and can immediately start acquiring data 451 by conducting a reverse genetics screen devised by the principal investigator and/or themselves. 452
Over time, these students can become confident enough to develop and plan their own projects. 453
In summary, this module is an excellent resource for instructors interested in conveying a 454
real-life science experience to their students and serves as an excellent opportunity for students to 455
gain the hands-on experience they need in order to pursue a career in biology. 456 457 RNAi efficiency and to avoid overcrowding/starvation, ~50 worms per plate will suffice. (Step 3) L1 494
larvae are grown until the L3/L4 larval stage and then mounted on 5% agarose pad slides (containing 495 sodium azide (anesthetic) and a drop of M9 buffer) for image acquisition. *Growth times will vary 496 based on temperature (see text for more details). (Step 4) Images are acquired and then analyzed 497
using Fiji/ImageJ to determine the mean fluorescence intensity. Results are briefly explained in the 498 lab report and submitted along with a publication quality figure with figure legend. 499 