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Human Practices

Table of contents:

Summary
The Handbook of Tested and Optimized Synthetic Biology Protocols for Amateurs
Rapid, Flexible, and Affordable Yeast Genome Engineering with BioBrick Standardization
Frugally Modernizing Synthetic Biology Techniques
1. Background: When Traditional Techniques Fail
2. DIY Gibson Assembly Kit

Summary:

Being an independent team of undergraduate students, we quickly learned that inexperience in synthetic biology and financial constraints limit the success of young teams. Thus, our project endeavoured to tackle these two obstacles. Thanks to our Virtual Protocol Donation Box, we analyzed, tested, and optimized a plethora of protocols from other successful iGEM teams. We compiled an easy-to-read protocol handbook designed specifically to assist new learners of synthetic biology. The handbook is equipped with software models designed by our dry lab to assist with primer design and yeast cloning. We allied with the University of Ottawa’s DEGREE program and invited students from various educational backgrounds and levels to test our protocols. We analyzed their performance and their feedback to ameliorate our protocols. Based on their comments, we filmed succinct videos to accompany each procedure. Our protocols are specifically designed to train amateurs of synthetic biology using cost-effective techniques. Our collaborators, who worked with higher order eukaryotes, recommended that we make the competition accessible to young synthetic biologists who are interested in working with eukaryotes. Consequently, we developed a library of BioBrick plasmid backbones that are simultaneously compatible for cloning in both E. coli and S. cerevisiae using the basic techniques explained in our protocols. To further overcome the financial hurdles of the competition while keeping up-to-date with modern cloning techniques—and to address common documented issues with traditional cloning that were noted by our collaborators and during the making of our yeast-compatible plasmid backbone library—, we developed a DIY Gibson Assembly kit to minimize the cost of cloning and provided an easy-to-read manual to make the kit. We provide the necessary enzyme sequences, cost-free, to the Registry of Standard Biological Parts.

1. The Handbook of Tested and Optimized Synthetic Biology Protocols for Amateurs

a. Background: Science, Finances, Politics

Technical complications of synthetic biology, in addition to financial constraints, are especially problematic for young scientists. We can speak from experience: iGEM is the first opportunity for all our team members to work in synthetic biology, and the learning curve was very steep. Not only we started the competition with hardly any knowledge of restriction enzymes, we had no funds to finance the team. To make matters worse, a new political party took power in our province and insisted on making our science life extra-difficult by cutting-down public education funds, which ultimately prevented our access to funding also. Although our prospects seemed bleak, given our inexperience and financial standing, we had a vision: make synthetic biology accessible to amateurs through simple, tested, optimized, and cost-effective training protocols. Our idea expanded to more than just a protocol handbook as we progressed through the project—continue reading for more details.

b. Virtual Protocol Donation Box

Thus, began a period of intense protocol review. We collected a plethora of tested protocols from a variety of sources. The first source was iGEM’s website. These protocols inspired us because they provided a brief introduction to explain the purpose of each procedure. Upon testing the protocols, however, we noticed that the protocols consistently recommended using less DNA, which assumed that the experimenter had a high experimental success rate, which was not our case. For example, in iGEM’s DNA extraction from a kit plate protocol, it recommends that only 1 µL of immersed DNA be transformed into home-made chemically competent E. coli. We found that volume to be very little and we had very limited growth on our antibiotic selection plate. Instead, we modified that protocol to indicate that all immersed DNA be used for the transformation. This enhanced growth on antibiotic selection agar plates.

Another source of protocols which we tested and optimized were commercial and non-commercial protocols which we found in our supervisor’s laboratory. For example, we tested BioBasic’s EZ-100 Column DNA Gel Extraction purification, and found very unpredictable yields, some of resulting in very successful cloning, and others resulting in very consistently unsuccessful cloning. We troubleshooted for temperature effects, ethanol contamination, and DNA shearing, but to no satisfying explanation for the unpredictable yields. Please see section 2b. RFC 10 Compatible Plasmid Backbones for further details and quantitative results. Therefore, we eliminated this protocol from our protocol compilation and we made no recommendation to use DNA gel extraction or heat-resistant restriction endonucleases because they were together prone to experimental failure leading to both time and monetary losses (Figure 2). We did, however, keep BioBasic’s miniprep plasmid extraction protocol because it demonstrated consistent high yields, albeit we did optimize the procedure to include longer incubation periods to enhance DNA quality and purity. Other protocol optimizations were as simple as recommending that experimenters use plastic pipette tips rather than wooden sticks for culture inoculations because wooden sticks absorbed over half of the inoculate during the overnight incubation, which reduced the yield of miniprep plasmid extractions (Figure 3).

**Figure 3.** Some protocol modifications were very simple. We modified Addgene’s inoculation protocol to recommend only the use of plastic pipettes to inoculate, because wooden sticks absorbed half of the culture during the overnight incubation.

A major source of protocols, however, was the international collaboration which Victoria Feng spearheaded: the Global Virtual Protocol Donation Box. Please visit our Collaborations page for further details on the teams that have contributed to the Donation Box. Fellow iGEMers from teams world-wide, who sympathized with our initiative, donated their own tested, optimized, and cost-effective synthetic biology protocols. VIT Vellore ligation protocol, for instance, provided a very easy to read step-by-step procedure for XbaI and SpeI cohesive-end ligation. We modified this protocol to make it universal to all ligations and also removed the segment about using gel extraction to purify the ligate. Instead, we recommended heat-inactivating the ligate and directly transforming it. This modification, not only increases the yield, it also reduces reduces the number of necessary steps prior to transformation. In another example, TU Dresden’s protocol for the preparation of chemically competent cells was very succinct, at the expense of missing some necessary details, such as the E. coli strain, the type of growth medium, the duration of growth, etc. We modified their protocol to include the missing materials, we included additional details regarding the incubation periods, the temperature of the room in which to work, and we eliminated the steps regarding storage in 10 % glycerol. We even compared the transformation efficiency of our chemically competent cells with that of NEB’s DH5α to ensure that our protocol is competitive and cheaper. QGEM’s protocols were highly beneficial, not for the training protocols, but for the DIY Gibson Assembly kit manual, because they provided insightful details regarding column purification, Western Blots, and Gibson Assembly. We compared their protocols to Dr. Rudner’s (our instructor) in the making of our own Gibson Assembly manual. Moreover, please note that we did not include all protocols from the Donation Box into our training protocols, because we deemed them too advanced; we wanted to focus on instilling the basic molecular biology protocols first. The Virtual Protocol Donation Box (Figure 4), however, remains open to all iGEM teams seeking experimental insight and troubleshooting alternatives.

After we compiled our tested and optimized protocols in a logical sequence into one manual, we validated our protocols by inviting synthetic biology amateurs from a variety of academic levels and backgrounds to test our protocols.

c. University of Ottawa DEGREE Program

As explained above, we developed a set of robust training protocols that could be easily understood and performed by undergraduate students with limited research experience. The training protocol was designed to replicate the workflow of developing new BioBricks. It included procedures such as DNA restriction digestion, DNA ligation, E. coli transformation, E. coli inoculation, E. coli colony PCR, plasmid DNA miniprep, and gel electrophoresis. As a part of our initiative to get more students interested in synthetic biology and to make synthetic biology more accessible, we collaborated with the University of Ottawa to host 4 students from the DEGREE (Discover and Engage Graduate Research Experience) program for three days. This program aimed to introduce undergraduate students to research performed at the University. These students, who had limited research experience, were provided the opportunity to perform our training protocols.

When the students first arrived, we provided them with some background information on iGEM, the purpose of each experiment, and the goal of the entire process – to create an RFP PSB1K3 BioBrick from CEN PSB1K3 and RFP PSB1A3.

Before each experiment, one of our team members, Emily Lam, performed the protocol to demonstrate the proper technique. Her results were also used as a positive control to compare the students’ results. After the demonstration, we asked the students to perform the protocol. Although Emily was present to oversee the students and provide guidance, all of the physical work (eg. pipetting) was performed by the students.

The first part of the experiment involved digesting CEN PSB1K3 and RFP PSB1A3 with EcoRI-HF and SpeI. Afterwards, the digested DNA was ligated and transformed on agar plates containing LB kanamycin. Each student also made a negative control, which consisted of E. coli with no transformed plasmid. We incubated the plates for 16 hours before viewing them. Every student’s negative control had no growth, which meant that there was no contamination. Because RFP provided colorimetric selection, the efficiency of each student’s transformation could be easily determined. The results of each student’s transformation are depicted in the following table:

All students, with the exception of one student, had at least one red colony present on their plate. We inoculated 3 colonies (or the maximum number of red colonies) from each individual’s plate in liquid LB kanamycin. For the student who did not have any red colonies, she continued her experiments using a backup plate that was prepared when the protocol was being tested. Each individual also performed a colony PCR of RFP. The results of the colony PCR can be seen in the following gels:

**Figure 8.** Ladder, B 0-2, pos, A 1-3, 0, pos

**Figure 9.** Ladder, D 0-3, pos, C 0-2, pos

**Figure 10.** Ladder, Emily 1-3, 0, positive

The gels show a single band around the 1000 base pair mark, which represents RFP. There was only one student (student A) whose colony PCR did not work. Given that her positive control also did not work, the PCR failure can likely be attributed to improper technique. We also ran everyone’s initial digestion product on a gel to assess whether or not the DNA was completely digested:

**Figure 11.** Ladder, Emily, D, C, B, A RFP, CEN

Emily and student D’s digestion products show complete digestion. Student B and C’s RFP PSB1A3, and student A’s CEN PSB1K3 did not digest completely. This likely explains the lack of red colonies on student A’s plates. After incubating the inoculations for 18 hours, the DNA was miniprepped and digested with EcoRI-HF and SpeI again to verify the BioBrick. The digestion products were run on a gel:

**Figure 12.** Ladder, Emily 1-3, D 1-3, C 1-2, B 1-2, A 1-3

The gel shows two bands at the correct length in every lane: the upper band represents PSB1K3, which is 2204 base pairs long. The lower band represents RFP, which is 1069 base pairs long. Two lanes show additional bands of different lengths, but the desired two bands are still present. These additional bands can be attributed to contamination. After the program, we sent each student a questionnaire to ask for feedback on our protocols.

d. Refined Protocol Handbook with Protocol Videos and Primer Designer

The feedback from the DEGREE visitors who tested our protocols was very helpful, and we used it to refine the protocols and make them clearer. For example, one student stated that although the protocols seemed somewhat abstract at first, they made sense once we put them to practice in the lab. Because someone with experience cannot always be present to provide assistance to students who are trying out the protocols, this gave us the idea to film video protocols to accompany our written protocols. We also noted that students struggled with the concept of primer synthesis. Therefore, Miléna Sokolowski, the dry lab member, designed a tool to assist with the making of the PCR primers. Please visit her software/dry lab page to learn more. The refined Handbook of Tested and Optimized Synthetic Biology Protocols for Amateurs can be found here. The series of video protocols can be found here: link.

2. Rapid, Flexible, and Affordable Yeast Genome Engineering with BioBrick Standardization

a. Background: Facilitating iGEMer’s Access to S. cerevisiae Cloning

While rummaging through iGEM’s websites in search for protocols, we noticed that the BioBrick backbones were limited to work with E. coli. There were very few resources for amateur synthetic biologists interested in working with higher-order eukaryotes. This could be due to a multitude of reasons, namely that cloning in eukaryotes is technically more difficult, time-consuming, and costly. Because our Handbook of Tested and Optimized Synthetic Biology Protocols for Amateurs also encompassed transformations in Saccharomyces cerevisiae, we believed that we should also work on making the competition more accessible to synthetic biologists interested in working with Saccharomyces cerevisiae. We felt doubly encouraged by Team UC Davis, who were also tackling the issue of the absence of eukaryotic representation in iGEM, save in mammalian cell lines. In brief, we develop a library of BioBrick plasmid backbones that are compatible for cloning in both E. coli and S. cerevisiae using the basic techniques explained in our protocols. The plasmid backbones adhere to the RFC 10 Standard and Type IIS Assembly and allow for the systematic and efficient cloning of a desired gene within target yeast chromosomal loci, Ade2, His3, Ade4, and Gal4 loci, via homologous recombination and are equipped with KanMX, NatMX, Ura3, and His3 yeast-selectable markers as well as RFP to enable colorimetric selection in E. coli. This library reduces the number of steps, and the amount of troubleshooting, preceding a yeast transformation. Setti Belhouari spearheaded this project by designing and constructing the RFC Standard 10 backbones, based on pSB1K3, that are compatible with homologous recombination in S. cerevisiae as part of her honours thesis project in the fourth year of her undergraduate studies. Setti Belhouari and Taylor Lanosky also expanded the plasmids by designing them such that they are also compatible with Type IIS Assembly. Taylor Lanosky constructed the remainder of the Type IIS Assembly yeast-compatible plasmid backbones.

b. RFC 10 Standard Yeast-Compatible Plasmid Backbones

In addition to complying with the BioBrickTM standard specifications, which enables the usage of the platform in the classic BioBrick Standard Assembly process, our goal was to update and design BioBrick plasmid backbones to enhance site-directed genomic modification via homologous recombination in S. cerevisiae, the most efficient method of genome modification in this organism. To do so, BioBrick backbone, pSB1K3, needed to include 1) upstream and downstream sequences that are homologous to various yeast loci allowing for homologous recombination, 2) an auxotrophic or an antibiotic selection marker that allows the identification of the yeast clones possessing the desired genome modification, and 3) a multiple cloning site that allowed the insertion of a desired coding sequence (Figure 13).

**Figure 13.** The proposed BioBrickTM Standard 10 update extends cloning from E. coli to S. cerevisiae. The updated plasmids facilitate site-directed homologous recombination in yeast because any desired insert, within the multiple cloning site, is flanked by a yeast-compatible selection marker as well as upstream and downstream target locus homologies.

Team:uOttawa/Human Practices