iGEM 2019 — Team Stanford

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# Team:Stanford/Notebook

Stanford iGEM Team Wiki

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![menu interface icon](https://static.igem.org/mediawiki/2019/b/b3/T--Stanford--menu.svg)

* [Project](https://2019.igem.org/Team%3AStanford/Description)

  [Overview](https://2019.igem.org/Team%3AStanford/Projects_Overview)
  [New Directed Evolution Method](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vitro)
  [DiCE In Vitro](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vitro)
  [DiCE In Vivo](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vivo)
  [Implementation](https://2019.igem.org/Team%3AStanford/PADE)
  [Novel Selection Schema](https://2019.igem.org/Team%3AStanford/PADE)
  [Novel Directed Evolution Application](https://2019.igem.org/Team%3AStanford/AcrIIA4)
  [Hardware](https://2019.igem.org/Team%3AStanford/Hardware)
  [Software](https://2019.igem.org/Team%3AStanford/Software)
  [Notebook](https://2019.igem.org/Team%3AStanford/Notebook)
* [Team](https://2019.igem.org/Team%3AStanford/Team)

  [Team Members](https://2019.igem.org/Team%3AStanford/Team)
  [Team Plus](https://2019.igem.org/Team%3AStanford/Team_Plus)
  [Collaborations](https://2019.igem.org/Team%3AStanford/Cell-Free_Interlab)
  [ourTXTL: Accessible Cell-Free](https://2019.igem.org/Team%3AStanford/Cell-Free_Interlab)
  [Attributions](https://2019.igem.org/Team%3AStanford/Attributions)
* [Parts](https://2019.igem.org/Team%3AStanford/Parts)

  [Basic Part](https://2019.igem.org/Team%3AStanford/Basic_Part)
  [Composite Part](https://2019.igem.org/Team%3AStanford/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3AStanford/Part_Collection)
* [Human](https://2019.igem.org/Team%3AStanford/Public_Engagement)

  [Safety at Every Step](https://2019.igem.org/Team%3AStanford/Safety)

  [Education and Engagement](https://2019.igem.org/Team%3AStanford/Public_Engagement)
  [High School Summer Internship Program](https://2019.igem.org/Team%3AStanford/Public_Engagement#hs)
  [Outreach](https://2019.igem.org/Team%3AStanford/Public_Engagement#or1)
* [Modeling](https://2019.igem.org/Team%3AStanford/Model)

  [Qbeta In Vivo Modeling](https://2019.igem.org/Team%3AStanford/Model#qbinvivo)
  [Promoter Strengths In Vitro](https://2019.igem.org/Team%3AStanford/Model#psinvitro)
  [Tuning Evolutionary Speed with Infection Reporters](https://2019.igem.org/Team%3AStanford/Model#ir)

**Interested in getting involved with Stanford iGEM 2020?  [Click here!](http:biome.bio/igem.html)**

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#### Contact Us

![marker-1 icon](https://static.igem.org/mediawiki/2019/4/4a/T--Stanford--marker.svg)
443 Via Ortega, Stanford, CA 94305

![mail icon](https://static.igem.org/mediawiki/2019/d/d6/T--Stanford--mail.svg)
igem2019stanford@list.stanford.edu

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© Stanford iGEM 2019

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# Notebook

Check out our weekly notes.

![](https://static.igem.org/mediawiki/2019/e/ec/T--Stanford--divider-3.svg)

## Our Lab History - beginning from June 14

1. Week One
   [Designing Experiments and Ordering Materials](https://docs.google.com/document/d/e/2PACX-1vSYu_bdNPufL80-ScZ3UeUuvJbowthPEsW-rKpPt5Bg77hbl6fJnA8acT6PWX4jwHT8bIam6LvK-868/pub)
2. Week Two
   [Transformation and Literature Review](https://docs.google.com/document/d/e/2PACX-1vQ_MuM6hVSpbJxocFFS5zLtAE4PKY1_sM54Wo2N3TcKjSUiqxYy1udUBvOTaJV9BZS0rn8m5lC0IICZ/pub)
3. Week Three
   [Cell-free Testing and Gibson Assembly](https://docs.google.com/document/d/e/2PACX-1vTm2FVbdZLSf0U47Brt0PSvsXMpgNqwQc8zi-xCXFrOOhw3JY5Zft1T3ihUv334geFyqGLr1pxxRdj6/pub)
4. Week Four
   [Anti-CRISPR (Acr) & myTXTL Cell-Free Experiments and Rational Protein Design](https://docs.google.com/document/d/e/2PACX-1vSA9gtZSxpWzqZmYYwOviXfhtJ5ijWTgXFAax0LUISy5xX20bjOkAbiZMCu0Q7LoWVxEGm709zFsR1h/pub)
5. Week Five
   [More Literature Review on and Troubleshooting with Qbeta](https://docs.google.com/document/d/e/2PACX-1vTvKI42xL_iQ88wGlflL-QevFWAwcgDGB_PuQS4Qw4AynpZ3mRVsVmj9DMBlpCOLSlgfIQGiJAi-OBc/pub)
6. Week Six
   [Qbeta in vivo Topo Cloning and AcrIIA4 in vivo Testing](https://docs.google.com/document/d/e/2PACX-1vQ457CN1-7_NJKpCXNiJkP-JYl7VIjpVI1tx-djbljXMUmzF-HhgmNec2VnK6maCdjTi4PDok65bVHz/pub)
7. Week Seven
   [More Qbeta in vitro Experiments and AcrIIA4 Testing](https://docs.google.com/document/d/e/2PACX-1vS1zWQ_p5wv-XItb19JJaDnJPtWjnWDKCzakYkk6QTjWm8_8VkybsY-ax5sQWtbiKQgyW1NqkB3xEtb/pub)
8. Week Eight
   [More Qbeta in vitro and Setting up Cell-Free Promoters System](https://docs.google.com/document/d/e/2PACX-1vS53USU_xrwj0LD9ME6EvWvmOc0IgX2zPsbsrhWSyoylDHSwb1yk3BtBhUzWJIl8JkeyAcE4JrF5UEF/pub)
9. Week Nine
   [Sequencing and pHEAT Testing](https://docs.google.com/document/d/e/2PACX-1vRTEnK3qevrNrWsqB7K010VV5LUCfLTdYWLnZcoW3ETcv6NzkoYxFZL5-_o3be_fpVJNMOqIqCHGBne/pub)
10. Week Ten
    [Infection Reporters Testing and More Sequencing](https://docs.google.com/document/d/e/2PACX-1vRTEnK3qevrNrWsqB7K010VV5LUCfLTdYWLnZcoW3ETcv6NzkoYxFZL5-_o3be_fpVJNMOqIqCHGBne/pub)
11. Week Eleven
    [Re-running Topo Cloning and More Gibson Assembly](https://docs.google.com/document/d/e/2PACX-1vRNP-5RdN0lF5JU7n0cdKS-mGqDoJLYfUxiDxLA0cbTrG3epJ_BnDZtUXrtjYoZX7vRtuzTKpq4lTDs/pub)
12. Week Twelve
    [More Sequencing and Troubleshooting](https://docs.google.com/document/d/e/2PACX-1vRed8h1eqeHPrWCoRHEAe2WhREIDWj7KtdIJTmTQH0EnLCNAG3fn9ziOkmdCpKTheffkPhD03o2sVMl/pub)
13. Week Thirteen
    [Qbeta in vivo - Amp Stop Codon and CmR Flip Testing](https://docs.google.com/document/d/e/2PACX-1vRQVxCzIK1z7l6q69jw6SeOCQLF4pNc4MWR9_9QUYFWoDNisURhpZo4JcBWqXF1nb8QDKB3kRUQwUbI/pub)


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# Team:Stanford/Experiments

Stanford iGEM Team Wiki

[![stanford-logo](https://static.igem.org/mediawiki/2019/2/22/T--Stanford--logo.png)
![](https://static.igem.org/mediawiki/2019/7/74/T--Stanford--logo_text.png)](https://2019.igem.org/Team%3AStanford)

![menu interface icon](https://static.igem.org/mediawiki/2019/b/b3/T--Stanford--menu.svg)

* [Project](https://2019.igem.org/Team%3AStanford/Description)

  [Overview](https://2019.igem.org/Team%3AStanford/Projects_Overview)
  [New Directed Evolution Method](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vitro)
  [DiCE In Vitro](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vitro)
  [DiCE In Vivo](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vivo)
  [Implementation](https://2019.igem.org/Team%3AStanford/PADE)
  [Novel Selection Schema](https://2019.igem.org/Team%3AStanford/PADE)
  [Novel Directed Evolution Application](https://2019.igem.org/Team%3AStanford/AcrIIA4)
  [Hardware](https://2019.igem.org/Team%3AStanford/Hardware)
  [Software](https://2019.igem.org/Team%3AStanford/Software)
  [Notebook](https://2019.igem.org/Team%3AStanford/Notebook)
* [Team](https://2019.igem.org/Team%3AStanford/Team)

  [Team Members](https://2019.igem.org/Team%3AStanford/Team)
  [Team Plus](https://2019.igem.org/Team%3AStanford/Team_Plus)
  [Collaborations](https://2019.igem.org/Team%3AStanford/Cell-Free_Interlab)
  [ourTXTL: Accessible Cell-Free](https://2019.igem.org/Team%3AStanford/Cell-Free_Interlab)
  [Attributions](https://2019.igem.org/Team%3AStanford/Attributions)
* [Parts](https://2019.igem.org/Team%3AStanford/Parts)

  [Basic Part](https://2019.igem.org/Team%3AStanford/Basic_Part)
  [Composite Part](https://2019.igem.org/Team%3AStanford/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3AStanford/Part_Collection)
* [Human](https://2019.igem.org/Team%3AStanford/Public_Engagement)

  [Safety at Every Step](https://2019.igem.org/Team%3AStanford/Safety)

  [Education and Engagement](https://2019.igem.org/Team%3AStanford/Public_Engagement)
  [High School Summer Internship Program](https://2019.igem.org/Team%3AStanford/Public_Engagement#hs)
  [Outreach](https://2019.igem.org/Team%3AStanford/Public_Engagement#or1)
* [Modeling](https://2019.igem.org/Team%3AStanford/Model)

  [Qbeta In Vivo Modeling](https://2019.igem.org/Team%3AStanford/Model#qbinvivo)
  [Promoter Strengths In Vitro](https://2019.igem.org/Team%3AStanford/Model#psinvitro)
  [Tuning Evolutionary Speed with Infection Reporters](https://2019.igem.org/Team%3AStanford/Model#ir)

**Interested in getting involved with Stanford iGEM 2020?  [Click here!](http:biome.bio/igem.html)**

![deco-blob-1 decoration](https://static.igem.org/mediawiki/2019/b/bb/T--Stanford--deco-blob-1.svg)

#### Contact Us

![marker-1 icon](https://static.igem.org/mediawiki/2019/4/4a/T--Stanford--marker.svg)
443 Via Ortega, Stanford, CA 94305

![mail icon](https://static.igem.org/mediawiki/2019/d/d6/T--Stanford--mail.svg)
igem2019stanford@list.stanford.edu

[![iGEM Logo](https://static.igem.org/mediawiki/2019/8/8c/T--Stanford--igem_logo_big.png)](http://tinyurl.com/stanfordigem)

[![BioE Logo](https://static.igem.org/mediawiki/2019/7/75/T--Stanford--bioe_logo.png)](https://bioengineering.stanford.edu)

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© Stanford iGEM 2019

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# Experiments

![](https://static.igem.org/mediawiki/2019/e/ec/T--Stanford--divider-3.svg)

### Experiments

[Overview](#overview)

[DiCE in vivo](#diceinvivo)

[DiCE in vitro](#diceinvitro)

[Novel Selection Schema](#nss)

[Novel DE Application](#nda)

#### Overview

While the optimal iGEM team size is 8 to 12 members, the average team contains only 5 full-time equivalents during the summer research months. We all know why this is—college students are busy. Whether it’s a limitation on volunteer hours or it’s a cap on the number of stipends a university can offer, the reality is that most teams are left wanting more person-power at the end of the day.

Another reality is that there are thousands of high school students that have had exposure to the fundamentals of molecular biology through classes, but who are still hoping to gain experience working with hands-on research. In fact, a recent survey showed that 77% of high schoolers  [“are either extremely or very interested in volunteering to gain work experience.”](https://www.nbcnews.com/news/education/interns-are-getting-younger-high-schoolers-hit-workplace-n45146)

**Seems like these two realities could perhaps combine synergistically?**

**……Our team thought so too.**

![Team Pic](https://static.igem.org/mediawiki/2019/c/c2/T--Stanford--cute-hschoolers.jpg)

Our interns after receiving their certificates of completion

Icon For Angle-double-left
[##### Design](https://2019.igem.org/Team%3AStanford/Design#diceinvivo)

[##### Results](https://2019.igem.org/Team%3AStanford/Results#diceinvivo)Icon For Angle-double-right

#### DiCE in vivo

**Experiment 1: Does Qβ Self-Replicate in E. coli?**

To summarize this first experiment, we tested for the ability of Qβ replicase to replicate sense mRNA, transcribed off a plasmid vector. In order to do so, we tested for the antisense strand of RNA, which could only form as a result of replication with Qβ. This was accomplished by, in short, synthesizing cDNA from extracted RNA after one 12-hour growth phase in a chloramphenicol dosage assay. Using primers, specific to either sense or antisense RNA strands to synthesize the cDNA, we could detect the presence of antisense RNA by running gels of both antisense and sense cDNA.

It is important to note that this experiment was run with the initial construct design, pRNA, as described in the design section above. For simplicity, the target gene is shown in green while the gene encoding for Qβ replicase is omitted as it is not necessary to mention for this experiment. The major steps taken for this experiment are depicted below.

![Team Pic](https://static.igem.org/mediawiki/2019/a/ab/T--Stanford--hgfgvh.jpg)

Upon extraction of RNA, using strand-specific primers, both sense and antisense strands are synthesized.

![Team Pic](https://static.igem.org/mediawiki/2019/8/8d/T--Stanford--njuyt656789.png)

In order to test for antisense RNA, detection of amplified anti-sense cDNAs had to be done. Using a regular PCR reaction, a series of positive and negative controls were done on both sense and antisense cDNA samples. The position of the internal primers serves as a positive control, testing for the presence of both sense and antisense RNAs as this primer pair falls entirely within the target gene region. As negative controls, flanking primers were used, shown above in blue and orange to show that internal primer detection was not a result of DNA contamination. This is because of the position of the flanking primers, covering both the nucleotide region on the mRNA and the DNA.

![Team Pic](ttps://2019.igem.org/wiki/images/2/26/T--Stanford--nbvhu678907.png)

Above are the expected results if Qβ replicase functions within the chassis organism, E. coli. A check mark indicates the presence of a strong band at the appropriate length (~600 pb) while an X indicates the lack thereof, while a - indicates the test was not done.

**Experiment 2: Can Qβ replicase Increase Antibiotic Resistance?**

While it does not substantiate the hypothesized mechanism depicted in the design section, it does suggest to our team that perfecting the DiCE system is possible within E. coli. It is important to note, however, that this experiment was done using pRNA, and not the optimized constructs. A more comprehensive set of experiments with the new construct designs, No Qβ and Qβ have been designed and are awaiting the success of our mRNA gene delivery protocol for E. coli.

To summarize the experiment, several rounds of chloramphenicol dosage assays were done to provide an increasing selective pressure to increase resistance by optimizing the gene (again, this was using the pRNA construct and, therefore, can’t substantiate this hypothesis without sequencing data). After each round, cells that had survived in the highest concentration of chloramphenicol were moved into the next assay at even higher concentrations.

**Experiment 3: Can mRNA Gene Delivery Be Accomplished in E. Coli and Maintained Qβ replicase?**

This experiment is still being troubleshooted due to the overwhelming lack of literature on the subject of mRNA gene deliver in E. coli. Currently, this experiment is being attempted under several varying conditions and is also awaiting new reagents and parts. It is crucial to note that this experiment is utilizing the new construct designs, No Qβ and Qβ. Our protocol for creating electrocompetent cells can be found [here](https://docs.google.com/document/d/1UZOMm8ogmct27m0iJakjEs69JuWW9WM_rGfC8TZ1q9E/edit?usp=sharing) in a Google doc as it is still under experimentation and not ready for publication on our website. Additionally, our electroporation protocol can be found [here](https://docs.google.com/document/d/1UZOMm8ogmct27m0iJakjEs69JuWW9WM_rGfC8TZ1q9E/edit?usp=sharing). Conditions tested are shown below:

![Team Pic](https://static.igem.org/mediawiki/2019/d/d1/T--Stanford--nbvh987607.png)

Icon For Angle-double-left
[##### Design](https://2019.igem.org/Team%3AStanford/Design#diceinvivo)

[##### Results](https://2019.igem.org/Team%3AStanford/Results#diceinvivo)Icon For Angle-double-right

#### DiCE in vitro

This section contains both resources for and details on the implementation of the following internship program components:

**** Program Outreach — Spreading the word
* Application Process
* Recommendations for Gaining Departmental Support***

* Safety
* Mentorship Structure
* Gathering and Implementing Program Feedback
* Closing Out the Program

#### *Program Outreach*

A common pitfall of outreach is to selectively reach out to those who are already well-connected to such opportunities. For our team, being situated in the Bay Area made this all the more pertinent: it would be all too easy to get ample applicants by simply contacting one or two private high schools with already well-established internship programs. We wanted to reach beyond this, however. Thus, we made it our goal to spread the word to people who may not otherwise have the opportunity to get engaged with lab research.

![Team Pic](https://static.igem.org/mediawiki/2019/0/0a/T--Stanford--email.png)

Email outreach to bioengineering graduate students

To accomplish this we:

1. Had each Stanford iGEM member contact 2-3 local biology teachers from a variety of high schools.
2. Had a departmental-wide announcement to graduate students advertising the program and encouraging them to forward our program information widely.
3. Did in-person outreach to spread the word to local high school students.

We put together and distributed the following informational packet to these different outlets. Note that this is a version prior to the expansion of the program to include minors:
<https://tinyurl.com/Stanford-iGEM2019-18program>

More recently, we improved the cover page to the following (see right). An editable version of this template can be downloaded here for teams interested in building upon this handout to use for their teams

![Team Pic](https://static.igem.org/mediawiki/2019/6/68/T--Stanford--poster-overview.png)

Updated iGEM Info Handout (template available to download)

#### *Application Process*

The goal of the application process was to identify candidates that have demonstrated interest in pursuing science beyond the classroom and that would be committed to the team for the duration of their internships. We emphasized that prior lab experience was not required or expected, and we encourage applicants to highlight other skills and experiences outside of the classroom that may have given them applicable skills to working with a team on a long-term project.

We designed an application process with two phases: a written application with a letter of recommendation from a teacher or mentor, followed by a face-to-face interview with two to three members of the iGEM team.

For the application we asked for a one-page cover letter addressing the following four questions:

1. Why would you like to work on the Stanford iGEM team this summer?
2. What level of commitment can you have this summer?
3. What prior experiences have you had that may be relevant to your experience this summer?
4. How have your prior experiences uniquely prepared you to contribute to iGEM?

They could submit this cover letter along with basic information at [this google form](https://docs.google.com/forms/d/e/1FAIpQLSfVkqJZRHPwlpkB5c2bsQeVvb1ONOVlm29PERNaJ5QDJjUB8w/viewform). As an additional resource, here are [email templates](https://docs.google.com/document/u/1/d/1m1-n7qjsy1r_411G20Gr8Ne_oNTwpGtTOx548c_xv9M/edit?usp=sharing) that our team used throughout the process. Although teams may need to modify these documents to to tailor them for their respective programs, we encourage teams to use them as starting points for their program and to reach out to us if they have any questions.

#### *Gaining Departmental Support*

Having minors work supervised exclusively by undergraduates in a lab with a variety of safety hazards creates significant liability. Thus, getting support for a program such as this is inherently political and can be a time-intensive and bureaucratic process—get started well-before summer to leave time for outreach and a substantive application process!

Our first recommendation is to reach out to immediate mentors that know the larger system within which your iGEM team is operating. For us, this was the teaching lab’s Lab Manager. She knew what entity would ultimately need to approve such a program (the BioE Department Chair), she had recommendations for putting together a proposal, she knew some of the safety training requirements and pointed us to resources for minor-specific requirements, and finally she could connect us to the intermediate faculty from which we would need to garner support before proceeding to the department chair.

Our [final proposal](https://drive.google.com/file/d/1EvmJWfUbTatp3vyFyJDwj3NrrbdVgOMv/view) was a modified version of the aforementioned informational handout, but with an additional letter of support from our lab manager.

![Image](https://static.igem.org/mediawiki/2019/a/af/T--Stanford--network.jpg)

Take the time to build relationships across your department

My final more personal recommendation here is to balance being both professional and personal as you build relationships with those across the department. Though it may at times seem like a bureaucratic web of rules, building mutual trust with all those you come across can go much further than you may expect.

#### *Safety*

Safety was at the forefront, tail-end, and just about every step of the internship program. Before entering lab we had a [comprehensive list](https://drive.google.com/file/d/1DSJO0t4fnpY53D0JUXiebJ1aIOQTxEN0/view) of required safety trainings and documentation to complete, as well as a detailed list of requirements while working in lab.

In addition to the usual lab safety standards, working with minors poses a few additional hurdles. While these are unique for each university, beyond parental consent and liability forms from parental guardians Stanford also required that all of the undergraduates on our team completed additional “Working with Minors” online trainings, got official background checks, and were fingerprinted.

In addition to the safety checklist, we [documented the expected types of lab work](https://drive.google.com/file/d/1XolyfKHjbivE38rVEqOJ5XE697RlJZoy/view) in which high school students may participate, and also outlined off-limit work (such as tissue culture and chemical room access). For all time in lab, we ensured high school students would always be paired with at least one undergraduate at all times.

![Image](https://static.igem.org/mediawiki/2019/d/d8/T--Stanford--safety-contract.png)

18+ Safety Contract

#### *Mentorship Structure*

We approached the mentorship structure with intention: we aimed to create a program that efficiently used both undergraduate and high school students’ time, that would expose interns to a variety of types of work, and that would allow interns to take ownership over part of the project.

We encouraged interns to commit to 6-8 weeks (four week minimum), working in lab 4 hours per day Monday to Friday. Our team quickly realized that it was most effective to have the mornings reserved for the undergrads to do logistical work, long-term project planning, and initial lab work, then to have the interns join us in lab at 1:00PM for the remainder of the day.

We set aside the first 2 weeks for interns to shadow a variety of the sub-projects our team were working on in order to maximize exposure to different lab techniques. We put together a document with descriptions and links to protocols for common lab techniques (including basic instructions for using Benchling), and had interns print and annotate the procedures.

![Image](https://static.igem.org/mediawiki/2019/6/6e/T--Stanford--mastering-protocol.png)

The interns' first assignment: Gaining familiarity with protocols fundamentals

Following this initial phase, we then decided the interns were ready to take on their own sub-project: AcrIIA4 Directed Evolution. With assistance when needed, we let them design primers, submit sequencing data, and ultimately plan their own lab work timeline.

For the final phase we helped the interns write up the content of their sub-project for the website (check out “AcrIIA4 Evolution”!). While recognizing that research is never complete, this final push was critical for pulling together the work they had completed thus far, for recognizing and contextualizing the motivations for the work they were doing, and for ultimately sharing their findings with the larger scientific community.

#### *Gathering and Implementing Program Feedback*

From the get-go, we were up front with the interns that the program was a novel one. We highly encouraged open communication and constructive feedback on both positive and negative aspects of the experience so that we could continue to improve.

To further encourage and formalize this process, we had interns fill out an anonymous satisfaction and feedback survey both midway and at the end of the program. We included an input field for a random number the intern could put so we could track performance longitudinally while still maintaining anonymity. We tracked some metrics quantitatively such as overall satisfaction and whether the interns felt their time was being used effectively, and we also gave space for more qualitative feedback such as “What aspects of the program would you like continued” and “Please provide 1 to 3 areas where we could improve to maximize your experience this summer.”

These survey results both helped us to make tweaks to the program midway through the summer, and it also gave us concrete areas to work on for next year (see data below!).

![Image](https://static.igem.org/mediawiki/2019/c/c2/T--Stanford--survey.png)

Interns feedback and satisfaction survey

#### *Closing Out the Program*

Though it may seem a small detail, we’re strong advocates of a fun last day :) On one of the last days of summer, we invited the three interns back for one last celebration of all the hard work we completed over the summer. We put together personalized certificates of completion for the high schoolers, we got some ice cream, and had a relaxing afternoon enjoying a picnic out on the oval.

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[##### Design](https://2019.igem.org/Team%3AStanford/Design#diceinvitro)

[##### Results](https://2019.igem.org/Team%3AStanford/Results#diceinvitro)Icon For Angle-double-right

#### Novel Selection Schema

Our interns stayed for 6 weeks, 7 weeks, and 11 weeks respectively. We were incredibly grateful for their choice to stay longer than required and to have their continued support with lab work and content creation for the website. By the end of the process they had become experts of PCR, gel extractions, mini-preps, cell-free protein expression, and much more. As they were able to gather expertise from working with a variety of lab members, by the end of the summer I personally would sometimes go to them for help with these protocols when I wanted tips and pointers.

Our survey data showed the following:

![Image](https://static.igem.org/mediawiki/2019/1/13/T--Stanford--survey-data.png)

Additionally, our short answers sections showed the following:

![Image](https://static.igem.org/mediawiki/2019/9/90/T--Stanford--survey-graph.png)

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[##### Design](https://2019.igem.org/Team%3AStanford/Design#nss)

[##### Results](https://2019.igem.org/Team%3AStanford/Results#nss)Icon For Angle-double-right

#### Novel DE Application

#### Experiments

Our first experiment was to test AcrIIA4 against both dCas9 and dxCas9 in cell free. We devised the following test groups:

![Image](https://static.igem.org/mediawiki/2019/f/f6/T--Stanford--ii.jpg)

We used the PURExpress® In Vitro Protein Synthesis Kit from NEB and ran our test groups in duplicates for 5 hours. During this time, we monitored fluorescence every ten minutes in our plate reader with excitation set to 520 nm and emission measurement at 610 nm.

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[##### Design](https://2019.igem.org/Team%3AStanford/Design#nda)

[##### Results](https://2019.igem.org/Team%3AStanford/Results#nda)Icon For Angle-double-right

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# Team:Stanford/Results

Stanford iGEM Team Wiki

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* [Project](https://2019.igem.org/Team%3AStanford/Description)

  [Overview](https://2019.igem.org/Team%3AStanford/Projects_Overview)
  [New Directed Evolution Method](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vitro)
  [DiCE In Vitro](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vitro)
  [DiCE In Vivo](https://2019.igem.org/Team%3AStanford/QB_DE_In_Vivo)
  [Implementation](https://2019.igem.org/Team%3AStanford/PADE)
  [Novel Selection Schema](https://2019.igem.org/Team%3AStanford/PADE)
  [Novel Directed Evolution Application](https://2019.igem.org/Team%3AStanford/AcrIIA4)
  [Hardware](https://2019.igem.org/Team%3AStanford/Hardware)
  [Software](https://2019.igem.org/Team%3AStanford/Software)
  [Notebook](https://2019.igem.org/Team%3AStanford/Notebook)
* [Team](https://2019.igem.org/Team%3AStanford/Team)

  [Team Members](https://2019.igem.org/Team%3AStanford/Team)
  [Team Plus](https://2019.igem.org/Team%3AStanford/Team_Plus)
  [Collaborations](https://2019.igem.org/Team%3AStanford/Cell-Free_Interlab)
  [ourTXTL: Accessible Cell-Free](https://2019.igem.org/Team%3AStanford/Cell-Free_Interlab)
  [Attributions](https://2019.igem.org/Team%3AStanford/Attributions)
* [Parts](https://2019.igem.org/Team%3AStanford/Parts)

  [Basic Part](https://2019.igem.org/Team%3AStanford/Basic_Part)
  [Composite Part](https://2019.igem.org/Team%3AStanford/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3AStanford/Part_Collection)
* [Human](https://2019.igem.org/Team%3AStanford/Public_Engagement)

  [Safety at Every Step](https://2019.igem.org/Team%3AStanford/Safety)

  [Education and Engagement](https://2019.igem.org/Team%3AStanford/Public_Engagement)
  [High School Summer Internship Program](https://2019.igem.org/Team%3AStanford/Public_Engagement#hs)
  [Outreach](https://2019.igem.org/Team%3AStanford/Public_Engagement#or1)
* [Modeling](https://2019.igem.org/Team%3AStanford/Model)

  [Qbeta In Vivo Modeling](https://2019.igem.org/Team%3AStanford/Model#qbinvivo)
  [Promoter Strengths In Vitro](https://2019.igem.org/Team%3AStanford/Model#psinvitro)
  [Tuning Evolutionary Speed with Infection Reporters](https://2019.igem.org/Team%3AStanford/Model#ir)

**Interested in getting involved with Stanford iGEM 2020?  [Click here!](http:biome.bio/igem.html)**

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#### Contact Us

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443 Via Ortega, Stanford, CA 94305

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igem2019stanford@list.stanford.edu

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© Stanford iGEM 2019

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![](https://static.igem.org/mediawiki/2019/7/7b/T--Stanford--results3.png)

# Results

![](https://static.igem.org/mediawiki/2019/e/ec/T--Stanford--divider-3.svg)

### Results

[Overview](#overview)

[DiCE in vivo](#diceinvivo)

[DiCE in vitro](#diceinvitro)

[Novel Selection Schema](#nss)

[Novel DE Application](#nda)

#### Overview

We developed Directed Chassis-agnostic Evolution, or DiCE, a novel, easy-to-implement selection-based directed evolution platform built off Qbeta replicase, an RNA-based RNA polymerase. We worked towards demonstrating DiCE’s ability to evolve proteins in both E. coli and cell-free environments.

For DiCE In Vivo, we 1) Demonstrated that Qbeta could replicate RNA in our in vivo system and thereby create the “anti-sense RNA” strand 2) Showed that Qbeta could “flip” an antisense chloramphenicol target gene and produce antibiotic resistance in E. coli, and 3) Modeled theoretical error frequency and distribution for our DiCE In Vivo system.

For DiCE in vitro we 1) Showed Qbeta-dependent RNA self-replication with a spinach aptamer 2) Showed Qbeta-dependent expression of a GFP target gene between MDVs and 3) Successfully implemented a full proof-of-concept DiCE In Vitro to evolve a T7 polymerase to have 400% increased transcriptional activity for a T3 promoter.

Furthermore, we generated standard selection schema compatible with PREDCEL (Heidelberg 2017) to expand the range of synthetic biological parts that can be created by any SSS.

We were able to make first steps towards monitoring evolutionary progress with the infection reporter that was developed and created a new framework for conducting M13-based evolutions.

Finally, we designed a selection schema to evolve a novel anti-CRISPR protein using our DiCE in vivo system.

Taken together, our work on SSS presents a foundational advance towards a future where part creation is easier, faster, and more accessible.

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[##### Experiments](https://2019.igem.org/Team%3AStanford/Experiments#overview)

#### DiCE in vivo

Currently, results have largely been impeded with the optimized constructs due to time and difficulty successfully establishing a working procedure of gene delivery into our electrocompetent E. coli. To the best of our knowledge, a single paper has been published within the last 40 years on the transfection of E. coli with mRNA by Akira Taketo, which has greatly limited the foundation from which we could work upon.8 This procedure is still under experimentation and being modified in order to move on to additionally planned experiments and proofs-of-concept in order to validate the hypothesized mechanism above.

Despite these current impediments to the in vivo portion of the DiCE project, data was gathered with the initial construct design. This data shows promise that Qβ replicase functions within bacteria and qualitatively suggests that mutant proteins were created for increased resistance to chloramphenicol. The latter, however, can’t be validated like the former due to a lack of sequencing data and inherent flaws of the initial construct design.

**Experiment 1: Results**

![Team Pic](https://static.igem.org/mediawiki/2019/4/4a/T--Stanford--nbvh9876507.png)

Above details the results of experiment 1. Shown above are three gels, with gel 2 testing for the presence of the target gene on each cDNA sample while gels 1 and 3 test for DNA contamination. All three gels use a positive control of the plasmid DNA, which holds the necessary nucleotide sequence for each primer pair to bind and amplify. On gel 2, the presence of the target gene is shown on both sense and antisense cDNAs as well as the positive control at the appropriate position on the ladder, suggesting the presence of antisense and sense mRNAs. On gel 1, contamination from plasmid DNA is analyzed by using a 3’ Flanking Primer pair, giving strong bands for the positive control but faint bands at inappropriate locations on the sense cDNA, indicating no plasmid contamination present in the sense cDNA sample, validating the positive results on gel 2. On gel 3, the positive control, the plasmid, shows a strong band for the 5’ Flanking Primer pair while the antisense cDNA sample has no appropriate bands present. This indicates that there was no plasmid contamination in the antisense cDNA sample, validating the positive results in gel 2, and strongly suggests that Qβ replicase function within our host organism.

**Experiment 2: Results:**

![Team Pic](https://static.igem.org/mediawiki/2019/7/75/T--Stanford--k8765hgfh.gif)

As shown above, chloramphenicol resistance appears to increase each subsequent generation. In the first generation (shown in blue) no cells survived past a 4X concentration of chloramphenicol. By generation 2 (shown in red) however, survival increased above what was anticipated and there was significant growth at an 8X concentration. The third generation (shown in yellow) increased as well, above the control group, which underwent the same rounds of evolution without the presence of Qβ replicase. This data implies that Qβ replicase was responsible for increased resistance in some way other than what the cell was already inherently capable of.

**Experiment 3: Results:**

We are still gathering data and troubleshooting. We will edit this page when publishable data is available. :)

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[##### Experiments](https://2019.igem.org/Team%3AStanford/Experiments#diceinvivo)

#### DiCE in vitro

If Qbeta was able to self replicate with the MDV regions as expected, then fluorescence should increase as a result of the spinach aptamer tag added to the Qbeta subunit RNA. Our results showed significant differences in fluorescent output between MDV-Qbeta-Spinach and mRFP-Spinach, with nearly a 500% increase in the fluorescence of the MDV-Qbeta-Spinach construct compared to the mRFP-Spinach background. This suggests that MDV-Qbeta-Spinach was operational in vitro in the used cell free lysate.

Testing MDV Region Impact on Replicability of Qbeta System

(IMAGE)

We hypothesized that flanking a given gene of interest, especially Qbeta, with the midivariants should make the whole sequence more conducive to replication and amplification by Qbeta. This was tested by comparing the fluorescent output of trials of MDV-Qbeta-Spinach construct in vitro with trials of a Qbeta-Spinach only construct, which lacked the MDV flanking regions. Run in triplicate, the results suggested a clear improvement of the MDV flanked Qbeta-Spinach over the control Qbeta-Spinach only, with no trial of the MDV-Qbeta-Spinach dipping below the highest performing trial of the Qbeta-Spinach. The differences in amplification of each construct suggested a ~30% improvement in the amplification rate with the MDV flanked construct versus the non-MDV flanked construct. MDV regions clearly play a critical role in Qbeta replicatibility, and can greatly enhance replication and amplification.

MDV Regions with Genes Beyond Qbeta

(IMAGE)

After demonstrating that the MDV regions allowed Qbeta self replication and were an important factor to the replicability of the Qbeta construct, the MDV regions were then applied to other target genes. The replicability of GFP RNA with and without MDV regions when exposed to Qbeta was determined in vitro, with a Qbeta construct featuring just the subunit as well as the full linked fusion protein. As shown in this data, GFP flanked by the MDV regions, in both the Qbeta full linker construct as well as with the subunit alone, were both shown to undergo replication with Qbeta in vitro, all present roughly ~2x above the background signal.

GFP Expression Inside Droplets:

(Image)

Droplets were clearly visible and their fluorescence was detectable under the microscope, showing that the cell free system and Qbeta replication/amplification worked successfully within the emulsification process. This means that the emulsification process is viable for creating the link between phenotype and genotype necessary for self selecting directed evolution, allowing us to proceed to the next step of developing an in vitro system to achieve this.

DiCE - Evolving a T7 RNA Polymerase to Bind T3 RNA Polymerase’s promoter In Vitro:

(Image)

Finally, once the feasibility of making non-Qbeta genes replicable and amplifiable by the MDV regions was demonstrated, the MDV regions were applied to the T7 Polymerase gene as an application of the Qbeta based DiCE method of directed evolution in vitro. By adding MDV regions to the T7 Polymerase, the T7 Polymerase was made replicable by Qbeta, which is a highly error-prone RNA transcriptase. This introduces mutations into each round of replication, which when put under selective pressure and linked to the replicability or availability of Qbeta in a system, should result in the accumulation of mutations that favor more well adapted candidates to the environment. In this system, the goal is the evolution of a T7 polymerase capable of binding to a T3 promoter region, and the selective pressure is introduced by linking Qbeta availability, which is responsible for replication, to binding and transcribing a T3 promoter. After 5 rounds of evolution, which involve passaging and expression inside cell free solution in increments, a ~400% increase was demonstrated in the rates at which T7 polymerase resulted in the expression of fluorescent reporter protein underneath T3 promoter regions with round 5 RNA compared to control non-evolved stock RNA for the polymerase. This suggests the power, speed, and accessibility of DiCE in vitro evolution as well as the usage of MDV regions for creating sequences replicable by Qbeta.

### Conclusion

With these experiments, we first initially were able to demonstrate the ability of Qbeta to replicate itself and undergo RNA amplification, with an increase in fluorescence of 500% compared to the baseline of the non-amplifying control. We were then able to demonstrate that the MDV regions increased the replicability of RNA sequences by Qbeta, showing that there was a ~30% increase in the fluorescence of the MDV-Qbeta-Spinach compared to the Qbeta-Spinach only construct. Next, we were able to show the ability of any arbitrary gene to be amplified and replicated by Qbeta through the addition of the MDV regions, with the MDV-GFP constructs which showed ~2x the activity of non-MDV GFP controls. Then we were able to demonstrate that we could create a link between genotype and phenotype that could be used for self selection in vitro via an emulsification process, which succeeded and was clearly visible with GFP constructs. After establishing all of these elements, we were able to conduct a T7 polymerase to T3 promoter binding affinity evolution and increase the activity of T7 polymerase with T3 promoter regions by ~400% compared to a non-evolved control. These results demonstrate that the DiCE In Vitro technique is a powerful and viable method of directed evolution. Featuring rapid prototyping, deep tunability, and a wide range of modifiable environmental variables, DiCE In Vitro demonstrates the ability to evolve constructs rapidly, effectively and without the barriers of traditional in vivo directed evolution techniques. More work needs to be done to test the power and limits of this new technique, but it is clear that the success of DiCE is beyond random.

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[##### Experiments](https://2019.igem.org/Team%3AStanford/Experiments#diceinvitro)

#### Novel Selection Schema

#### *Infection Reporter Characterization*

Due to the similarity between each of the infection reporters, we expected both of them to give high expression when infected and low expression if not. However, when both were expressed in a plasmid with a CloDF13 origin of replication (copy number 20-4014), they both exhibited a high variability in expression, with some infected cells giving no fluorescence levels above baseline, others giving slight increases in fluorescence, and still others giving large increases in fluorescence.

![Image](https://static.igem.org/mediawiki/2019/b/b3/T--Stanford--results41.png)

Figure 4.

A single colony of either TG-1 and S2060 cells with infection reporters (parts BBa\_K3258045 and BBa\_K3258006) driven by the respective biobrick parts in a BBa\_K3258044 backbone was picked and immediately infected with 2•107 pfu. Cells were then grown in LB with 35 µg/mL chloramphenicol and 25 mM glucose for 20 hours shaking at 37ºC.

Based on the results in the figure above, we decided to focus on characterizing Ppsp in S2060 cells. To potentially elucidate the reasons for high expression variability, we hypothesized that the promoter was differentially active at different phases of growth. Thus, we decided to infect the cells at various stages in the E. coli growth cycle as shown below:

![Image](https://static.igem.org/mediawiki/2019/a/a0/T--Stanford--results51.png)

Figure 5.

An overnight culture of S2060 cells with infection reporters (parts BBa\_K3258045 and BBa\_K3258006) driven by the respective biobrick parts in a BBa\_K3258044 backbone was picked and grown to saturation overnight. Cells were diluted 1:100 into fresh LB with 35 µg/mL chloramphenicol and 25 mM glucose, grown at 37ºC, and infected at the indicated time points. After 8 hours, the cells were grown for an additional 24 hours and their fluorescence and OD600 values measured.

Based on the results above, we decided to stick with using part BBa\_K2832003 as the promoter for our infection reporter as opposed to the new BBa\_K2832000.

Despite our best efforts, we were unable to conduct a successful evolution of allowing the T7 polymerase to bind to the T3 promoter using PREDCEL+ due to ambiguous results as a result of our passaging experiments. We will continue to troubleshoot these issues after iGEM. However, we had initial early signs of success when we saw plaques on plates transformed with all plasmids necessary for the evolution (SP, HP, and APMP).

![Image](https://static.igem.org/mediawiki/2019/d/d2/T--Stanford--figure62.jpg)

Figure 6. S2060 cells with the APMP (BBa\_K3258046) and HP (BBa\_K3258008) were made competent and transformed with the SP plasmid (BBa\_K3258014) and plated on LB agar plates with 50 µg/mL kanamycin, 35 µg/mL chloramphenicol, and 1mM rhamnose and grown at 37ºC. Plaques were visualized by eye.

Uninfected cells were diluted 1:5000 into LB with 50 µg/mL kanamycin, 35 µg/mL chloramphenicol, and 25 mM arabinose with variable amounts of rhamnose (10 mM to no rhamnose based on plaques assays) along with 0.5 µL of infected cells from a previous round and cultured in 50 mL conical tubes shaking at 37ºC overnight. Normalized fluorescence (ex. 488 nm, em. 530 nm) measurements were taken to monitor infection, but proved unreliable when compared to plaque assays or rolling circle amplification followed by PCR amplification (RCA-PCR).

After 6 rounds of evolution with the final round growing without rhamnose supplementation, an RCA-PCR was performed and a band detected corresponding to a 500 bp region of T7 where mutations were expected. However, sequencing results returned the consensus T7 polymerase sequence corresponding to that sequence.

We will continue to troubleshoot this evolution before and after the Jamboree and, once validated, move to test our biosensor selection schema.

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[##### Experiments](https://2019.igem.org/Team%3AStanford/Experiments#nss)

#### Novel DE Application

#### Results

Because cell free is expensive, we started with testing just the dCas9 groups first to demonstrate that all of our individual components were functioning as expected. Following a few suboptimal trials where we struggled with bubbles in the plate reader, we obtained the following data.

![Image](https://static.igem.org/mediawiki/2019/0/00/T--Stanford--jj.jpg)

Our positive and negative controls gave the expected results: our RFP fragment fluoresced, and our H2O negative control had minimal background. We saw marked lower expression for one our RFP positive controls, perhaps due to pipetting error.

Our RFP + scrambled sgRNA + dCas9 group fluoresced as expected (because the sgRNA variable region is scrambled and therefore unable to guide the dCas9 to the RFP gene for knockdown). What’s more, our trial with RR1 and dCas9 led to significant decrease. However, these initial results did not demonstrate AcrIIA4-dependent return of RFP expression: both our RFP+RR1+dCas9 + either dead AcrIIA4 or normal AcrIIA4 groups behaved the same and did not fluoresce.

After rerunning these AcrIIA4 groups specifically and seeing similar results, we sequence verified to confirm that we had successfully added the T7 promoter, RBS, and T7 terminator to our linear fragment. Because this sequencing data checked out, we were left with inconclusive results for AcrIIA4. We had two hypotheses as to why this might have been:

In Vitro1. Occasionally proteins that are functional in vivo are not able to fold properly in cell free extract. Though this is uncommon it is possible that AcrIIA4 might have been one of these proteins.
2. Perhaps more likely, the AcrIIA4 was struggling to express because it’s DNA sequence was mammalian codon optimized instead of bacterial. Though it is often not a problem to express mammalian optimized vectors in bacteria or bacteria extract, it can sometimes lead to far lower expression levels that could be responsible for the observed lack of effectiveness.

At this point in time, we unfortunately realized we were running low on our PURExpress kit. The cost to purchase more reactions was significantly high and we were still quite far from answering our initial question, so we decided to pivot our energy to other subprojects.

Instead, we shifted our focus to implementing a cost-effective protocol to develop our own cell-free extract, which we called ourTXTL. Check out this process here! Our hope was to return to cell free testing once we finalized this protocol, but unfortunately we only got successful results with ourTXTL later in the summer at which point it was too late to pick up this project again!

We did, however, carefully design a three plasmid system that could effectively evolve AcrIIA4 to be more effective against dxCas9 using the DiCE in vivo evolutionary system. See below.

#### Conclusion

While we were not able to conclude whether AcrIIA4 is effective against dxCas9 this summer, we do indeed think this project is worth pursuing in the future. Especially given that cell free expression is far more accessible given the ourTXTL process we worked on this summer, we hope to return to additional testing following the Jamboree.

Furthermore, we went ahead and envisioned the following DiCE in vivo selection schema to evolve AcrIIA4 to be better at inhibiting dxCas9. Once we get the answer to our initial question of AcrIIA4’s current effectiveness against dxCas9, we can pretty easily clone the following system by simply swapping in AcrIIA4 to be between the Qbeta binding MDVs with restriction enzymes, and changing the 20 base pair variable region of the sgRNA to block CmR expression. See our video for a more detailed explanation of our AcrIIA4 DiCE selection system!

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[##### Experiments](https://2019.igem.org/Team%3AStanford/Experiments#diceinvitro)