iGEM 2021 — Team Virginia

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Virginia/Notebook

Manifold

![](https://static.igem.org/mediawiki/2021/3/35/T--Virginia--images--loading.gif)

![](https://static.igem.org/mediawiki/2021/e/e0/T--Virginia--LogoHover--1.svg)

MANIFOLD

Synthetic Nanoreactors

[HOME](https://2021.igem.org/Team%3AVirginia)

[Main](https://2021.igem.org/Team%3AVirginia)

[PROJECT](#project)

[Description](https://2021.igem.org/Team%3AVirginia/Description)
[Experiments](https://2021.igem.org/Team%3AVirginia/Experiments)[Results](https://2021.igem.org/Team%3AVirginia/Results)
[Modeling](https://2021.igem.org/Team%3AVirginia/Model)
[Engineering](https://2021.igem.org/Team%3AVirginia/Engineering)[Notebook](https://2021.igem.org/Team%3AVirginia/Notebook)

[OUTREACH](#outreach)

[HP Introduction](https://2021.igem.org/Team%3AVirginia/Human_Practices)
[Identify the Problem](https://2021.igem.org/Team%3AVirginia/ItP)
[Imagine Solutions](https://2021.igem.org/Team%3AVirginia/IS)
[Gain Insight](https://2021.igem.org/Team%3AVirginia/GI)
[Implement the Solution](https://2021.igem.org/Team%3AVirginia/ItS)
[Adjust the Project](https://2021.igem.org/Team%3AVirginia/AtP)
[Public Engagement](https://2021.igem.org/Team%3AVirginia/Public_engagement)
[Collaborations](https://2021.igem.org/Team%3AVirginia/Collaborations)
[Contributions](https://2021.igem.org/Team%3AVirginia/Contribution)
[Scientific Communications](https://2021.igem.org/Team%3AVirginia/Entrepreneurship)

[TEAM](#team)

[Members](https://2021.igem.org/Team%3AVirginia/Members)
[Attributions](https://2021.igem.org/Team%3AVirginia/Attributions)

[AWARDS](#awards)

[Medals](https://2021.igem.org/Team%3AVirginia/Medals)

[RESOURCES](#about)

[Sources](https://2021.igem.org/Team%3AVirginia/Papers)
[Protocols](https://2021.igem.org/Team%3AVirginia/Protocols)

![](https://static.igem.org/mediawiki/2021/3/36/T--Virginia--images--designbackground.png)

#000000

Lab Notebook

Notebook Records

Raw Notes

Introduction

The following files are raw lab notes from various members of the UVA IGEM 2021 Team over the course of many months of hard work. These documents show the extreme level of planning and attention of detail required when developing our project  MANIFOLD .

February

[Collin 02-05-2021](https://static.igem.org/mediawiki/2021/5/59/T--Virginia--Lab_Notes--Lab_Notes--Collin_02-05-2021.pdf)

[Collin 02-13-2021](https://static.igem.org/mediawiki/2021/3/33/T--Virginia--Lab_Notes--Lab_Notes--Collin_02-13-2021.pdf)

March

[Collin 03-01-2021](https://static.igem.org/mediawiki/2021/9/9d/T--Virginia--Lab_Notes--Lab_Notes--Collin_03-01-2021.pdf)

[Collin 03-03-2021](https://static.igem.org/mediawiki/2021/9/91/T--Virginia--Lab_Notes--Lab_Notes--Collin_03-03-2021.pdf)

April

[Collin 04-08-2021](https://static.igem.org/mediawiki/2021/d/dd/T--Virginia--Lab_Notes--Lab_Notes--Collin_04-08-2021.pdf)

[Collin 04-09-2021](https://static.igem.org/mediawiki/2021/8/80/T--Virginia--Lab_Notes--Lab_Notes--Collin_04-09-2021.pdf)

[Collin 04-10-2021](https://static.igem.org/mediawiki/2021/f/fc/T--Virginia--Lab_Notes--Lab_Notes--Collin_04-10-2021.pdf)

[Collin 04-14-2021](https://static.igem.org/mediawiki/2021/3/3b/T--Virginia--Lab_Notes--Lab_Notes--Collin_04-14-2021.pdf)

[Collin 04-15-2021](https://static.igem.org/mediawiki/2021/f/f9/T--Virginia--Lab_Notes--Lab_Notes--Collin_04-15-2021.pdf)

[Collin 04-16-2021](https://static.igem.org/mediawiki/2021/2/22/T--Virginia--Lab_Notes--Lab_Notes--Collin_04-16-2021.pdf)

[Collin 04-17-2021](https://static.igem.org/mediawiki/2021/2/25/T--Virginia--Lab_Notes--Lab_Notes--Collin_04-17-2021.pdf)

May

[Collin 05-14-2021](https://static.igem.org/mediawiki/2021/9/92/T--Virginia--Lab_Notes--Lab_Notes--Collin_05-14-2021.pdf)

[Collin 05-17-2021](https://static.igem.org/mediawiki/2021/b/b9/T--Virginia--Lab_Notes--Lab_Notes--Collin_05-17-2021.pdf)

[Collin 05-27-2021](https://static.igem.org/mediawiki/2021/8/88/T--Virginia--Lab_Notes--Lab_Notes--Collin_05-27-2021.pdf)

[Collin 05-31-2021](https://static.igem.org/mediawiki/2021/8/81/T--Virginia--Lab_Notes--Lab_Notes--Collin_05-31-2021.pdf)

June

[Annie 06-03-2021](https://static.igem.org/mediawiki/2021/f/fb/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-03-2021.pdf)

[Annie 06-04-2021](https://static.igem.org/mediawiki/2021/7/77/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-04-2021.pdf)

[Annie 06-08-2021](https://static.igem.org/mediawiki/2021/a/ac/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-08-2021.pdf)

[Annie 06-09-2021](https://static.igem.org/mediawiki/2021/7/7f/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-09-2021.pdf)

[Annie 06-10-2021](https://static.igem.org/mediawiki/2021/8/84/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-10-2021.pdf)

[Annie 06-11-2021](https://static.igem.org/mediawiki/2021/4/46/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-11-2021.pdf)

[Annie 06-15-2021](https://static.igem.org/mediawiki/2021/a/aa/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-15-2021.pdf)

[Annie 06-16-2021](https://static.igem.org/mediawiki/2021/c/ce/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-16-2021.pdf)

[Annie 06-18-2021](https://static.igem.org/mediawiki/2021/e/e6/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-18-2021.pdf)

[Annie 06-22-2021](https://static.igem.org/mediawiki/2021/e/ef/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-22-2021.pdf)

[Annie 06-23-2021](https://static.igem.org/mediawiki/2021/3/3e/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-23-2021.pdf)

[Annie 06-24-2021](https://static.igem.org/mediawiki/2021/e/ed/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-24-2021.pdf)

[Annie 06-25-2021](https://static.igem.org/mediawiki/2021/1/16/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-25-2021.pdf)

[Annie 06-26-2021](https://static.igem.org/mediawiki/2021/6/67/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-26-2021.pdf)

[Annie 06-29-2021](https://static.igem.org/mediawiki/2021/c/c4/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-29-2021.pdf)

[Annie 06-30-2021](https://static.igem.org/mediawiki/2021/5/59/T--Virginia--Lab_Notes--Lab_Notes--Annie_06-30-2021.pdf)

[Collin 06-01-2021](https://static.igem.org/mediawiki/2021/f/fc/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-01-2021.pdf)

[Collin 06-02-2021](https://static.igem.org/mediawiki/2021/6/68/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-02-2021.pdf)

[Collin 06-03-2021](https://static.igem.org/mediawiki/2021/f/f0/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-03-2021.pdf)

[Collin 06-08-2021](https://static.igem.org/mediawiki/2021/4/46/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-08-2021.pdf)

[Collin 06-09-2021](https://static.igem.org/mediawiki/2021/f/f2/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-09-2021.pdf)

[Collin 06-10-2021](https://static.igem.org/mediawiki/2021/d/d2/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-10-2021.pdf)

[Collin 06-12-2021](https://static.igem.org/mediawiki/2021/e/e9/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-12-2021.pdf)

[Collin 06-13-2021](https://static.igem.org/mediawiki/2021/2/26/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-13-2021.pdf)

[Collin 06-19-2021](https://static.igem.org/mediawiki/2021/b/b2/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-19-2021.pdf)

[Collin 06-21-2021](https://static.igem.org/mediawiki/2021/d/d3/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-21-2021.pdf)

[Collin 06-22-2021](https://static.igem.org/mediawiki/2021/e/ef/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-22-2021.pdf)

[Collin 06-23-2021](https://static.igem.org/mediawiki/2021/2/2c/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-23-2021.pdf)

[Collin 06-25-2021](https://static.igem.org/mediawiki/2021/9/9e/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-25-2021.pdf)

[Collin 06-26-2021](https://static.igem.org/mediawiki/2021/d/da/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-26-2021.pdf)

[Collin 06-28-2021](https://static.igem.org/mediawiki/2021/4/44/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-28-2021.pdf)

[Collin 06-30-2021](https://static.igem.org/mediawiki/2021/a/a6/T--Virginia--Lab_Notes--Lab_Notes--Collin_06-30-2021.pdf)

[Maria 06-02-2021](https://static.igem.org/mediawiki/2021/6/65/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-02-2021.pdf)

[Maria 06-03-2021](https://static.igem.org/mediawiki/2021/e/ec/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-03-2021.pdf)

[Maria 06-04-2021](https://static.igem.org/mediawiki/2021/2/2a/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-04-2021.pdf)

[Maria 06-05-2021](https://static.igem.org/mediawiki/2021/0/04/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-05-2021.pdf)

[Maria 06-06-2021(1)](https://static.igem.org/mediawiki/2021/7/7d/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-06-2021%281%29.pdf)

[Maria 06-06-2021](https://static.igem.org/mediawiki/2021/1/12/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-06-2021.pdf)

[Maria 06-07-2021](https://static.igem.org/mediawiki/2021/8/83/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-07-2021.pdf)

[Maria 06-08-2021](https://static.igem.org/mediawiki/2021/e/e8/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-08-2021.pdf)

[Maria 06-10-2021(1)](https://static.igem.org/mediawiki/2021/1/1e/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-10-2021%281%29.pdf)

[Maria 06-10-2021(2)](https://static.igem.org/mediawiki/2021/9/91/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-10-2021%282%29.pdf)

[Maria 06-10-2021(3)](https://static.igem.org/mediawiki/2021/3/39/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-10-2021%283%29.pdf)

[Maria 06-10-2021](https://static.igem.org/mediawiki/2021/d/d3/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-10-2021.pdf)

[Maria 06-11-2021](https://static.igem.org/mediawiki/2021/b/b5/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-11-2021.pdf)

[Maria 06-14-2021](https://static.igem.org/mediawiki/2021/3/34/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-14-2021.pdf)

[Maria 06-15-2021](https://static.igem.org/mediawiki/2021/2/23/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-15-2021.pdf)

[Maria 06-19-2021](https://static.igem.org/mediawiki/2021/9/99/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-19-2021.pdf)

[Maria 06-21-2021](https://static.igem.org/mediawiki/2021/6/6d/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-21-2021.pdf)

[Maria 06-25-2021](https://static.igem.org/mediawiki/2021/d/d9/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-25-2021.pdf)

[Maria 06-26-2021](https://static.igem.org/mediawiki/2021/c/c8/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-26-2021.pdf)

[Maria 06-30-2021(1)](https://static.igem.org/mediawiki/2021/a/af/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-30-2021%281%29.pdf)

[Maria 06-30-2021(2)](https://static.igem.org/mediawiki/2021/9/91/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-30-2021%282%29.pdf)

[Maria 06-30-2021](https://static.igem.org/mediawiki/2021/2/28/T--Virginia--Lab_Notes--Lab_Notes--Maria_06-30-2021.pdf)

July

[Allison 07-09-2021(2)](https://static.igem.org/mediawiki/2021/3/3d/T--Virginia--Lab_Notes--Lab_Notes--Allison_07-09-2021%282%29.pdf)

[Allison 07-09-2021(1)](https://static.igem.org/mediawiki/2021/b/b3/T--Virginia--Lab_Notes--Lab_Notes--Allison_07-09-2021%281%29.pdf)

[Allison 07-09-2021(3)](https://static.igem.org/mediawiki/2021/7/7e/T--Virginia--Lab_Notes--Lab_Notes--Allison_07-09-2021%283%29.pdf)

[Allison 07-09-2021(4)](https://static.igem.org/mediawiki/2021/7/71/T--Virginia--Lab_Notes--Lab_Notes--Allison_07-09-2021%284%29.pdf)

[Allison 07-09-2021(5)](https://static.igem.org/mediawiki/2021/c/c5/T--Virginia--Lab_Notes--Lab_Notes--Allison_07-09-2021%285%29.pdf)

[Allison 07-09-2021](https://static.igem.org/mediawiki/2021/2/2d/T--Virginia--Lab_Notes--Lab_Notes--Allison_07-09-2021.pdf)

[Allison 07-13-2021](assets/Lab%20Notes/Lab%20Notes/Allison%2007-13-2021.pdf)

[Collin 07-01-2021](https://static.igem.org/mediawiki/2021/e/ea/T--Virginia--Lab_Notes--Lab_Notes--Collin_07-01-2021.pdf)

[Collin 07-05-2021](https://static.igem.org/mediawiki/2021/1/14/T--Virginia--Lab_Notes--Lab_Notes--Collin_07-05-2021.pdf)

[Collin 07-07-2021](https://static.igem.org/mediawiki/2021/a/a8/T--Virginia--Lab_Notes--Lab_Notes--Collin_07-07-2021.pdf)

[Collin 07-10-2021](https://static.igem.org/mediawiki/2021/b/b3/T--Virginia--Lab_Notes--Lab_Notes--Collin_07-10-2021.pdf)

[David 07-06-2021](https://static.igem.org/mediawiki/2021/8/8c/T--Virginia--Lab_Notes--Lab_Notes--David_07-06-2021.pdf)

[Maria 07-01-2021](https://static.igem.org/mediawiki/2021/5/55/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-01-2021.pdf)

[Maria 07-02-2021](https://static.igem.org/mediawiki/2021/9/94/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-02-2021.pdf)

[Maria 07-12-2021(1)](https://static.igem.org/mediawiki/2021/1/1c/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-12-2021%281%29.pdf)

[Maria 07-12-2021](https://static.igem.org/mediawiki/2021/f/f4/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-12-2021.pdf)

[Maria 07-14-2021(1)](https://static.igem.org/mediawiki/2021/a/a4/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-14-2021%281%29.pdf)

[Maria 07-14-2021](https://static.igem.org/mediawiki/2021/3/33/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-14-2021.pdf)

[Maria 07-15-2021](https://static.igem.org/mediawiki/2021/5/5e/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-15-2021.pdf)

[Maria 07-16-2021](https://static.igem.org/mediawiki/2021/8/84/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-16-2021.pdf)

[Maria 07-21-2021(1)](https://static.igem.org/mediawiki/2021/5/5c/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-21-2021%281%29.pdf)

[Maria 07-21-2021](https://static.igem.org/mediawiki/2021/0/04/T--Virginia--Lab_Notes--Lab_Notes--Maria_07-21-2021.pdf)

August

[Allison 08-06-2021](https://static.igem.org/mediawiki/2021/2/21/T--Virginia--Lab_Notes--Lab_Notes--Allison_08-06-2021.pdf)

[Allison 08-09-2021](https://static.igem.org/mediawiki/2021/9/95/T--Virginia--Lab_Notes--Lab_Notes--Allison_08-09-2021.pdf)

[Allison 08-10-2021](https://static.igem.org/mediawiki/2021/9/98/T--Virginia--Lab_Notes--Lab_Notes--Allison_08-10-2021.pdf)

September

[Allison 09-26-2021](https://static.igem.org/mediawiki/2021/0/06/T--Virginia--Lab_Notes--Lab_Notes--Allison_09-26-2021.pdf)

[David 09-26-2021(1)](https://static.igem.org/mediawiki/2021/e/e9/T--Virginia--Lab_Notes--Lab_Notes--David_09-26-2021%281%29.pdf)

[David 09-26-2021](https://static.igem.org/mediawiki/2021/8/85/T--Virginia--Lab_Notes--Lab_Notes--David_09-26-2021.pdf)

[David 09-29-2021](https://static.igem.org/mediawiki/2021/8/89/T--Virginia--Lab_Notes--Lab_Notes--David_09-29-2021.pdf)

October

[David 10-14-2021](https://static.igem.org/mediawiki/2021/2/21/T--Virginia--Lab_Notes--Lab_Notes--David_10-14-2021.pdf)

[David 10-16-2021](https://static.igem.org/mediawiki/2021/0/09/T--Virginia--Lab_Notes--Lab_Notes--David_10-16-2021.pdf)

![](https://static.igem.org/mediawiki/2021/8/81/T--Virginia--logo-up-frames--1.svg)

![](https://static.igem.org/mediawiki/2021/0/06/T--Virginia--images--uvawhite.png)
![](https://static.igem.org/mediawiki/2021/6/67/T--Virginia--images--logotextwhite.svg)

Manifold

Home

Project

Parts

Outreach

Team

Awards

Resources

Contact Us

Chemistry Building
485 McCormick Rd
CHARLOTTESVILLE, VA 22903

uvaigem@gmail.com

Sponsors

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![](https://static.igem.org/mediawiki/2021/3/35/T--Virginia--LogoHover--10.svg)
![](https://static.igem.org/mediawiki/2021/8/81/T--Virginia--LogoHover--11.svg)
![](https://static.igem.org/mediawiki/2021/f/fb/T--Virginia--LogoHover--12.svg)
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![](https://static.igem.org/mediawiki/2021/9/92/T--Virginia--LogoHover--15.svg)
![](https://static.igem.org/mediawiki/2021/4/43/T--Virginia--LogoHover--16.svg)
![](https://static.igem.org/mediawiki/2021/3/33/T--Virginia--LogoHover--17.svg)
![](https://static.igem.org/mediawiki/2021/8/8b/T--Virginia--LogoHover--18.svg)
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![](https://static.igem.org/mediawiki/2021/0/00/T--Virginia--LogoHover--21.svg)
![](https://static.igem.org/mediawiki/2021/8/81/T--Virginia--logo-up-frames--1.svg)
![](https://static.igem.org/mediawiki/2021/1/17/T--Virginia--logo-up-frames--2.svg)
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# Team:Virginia/Engineering

Manifold

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MANIFOLD

Synthetic Nanoreactors

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[TEAM](#team)

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Engineering

Index:

Engineering Design Cycle

Design of the Manifold System

Designing the Manifold system went through numerous different phases during the initial planning of the system's parts. From modeling to design to experimentation and verification, the design of each part was verified, judged, and constructed. We can’t pretend that our careful planning made this process go off without a hitch; in fact, the trial and error of the engineering design cycle was a necessary part of improving our project.

Trial and Error

The best example of our engineering design cycle is our reverse transcriptase (RT) part. RT is necessary to transcribe the RNA of our scaffold genes back into DNA, which can then bind to our zinc finger-fusion enzymes. We originally planned to assemble our RT part from standard plasmids that could be ordered from places like Addgene, thus saving money on gene synthesis. Once these standard plasmids were obtained, we originally hoped to assemble the entire system through a series of PCR reactions and ligations.

When designing our DNA scaffolds, we learned from the paper Genetic Encoding of DNA Nanostructures and their Self Assembly in Living Bacteria

[1]Elbaz, J., Yin, P. & Voigt, C. Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nat Commun 7, 11179 (2016). https://doi.org/10.1038/ncomms11179

that a combination of HIV-RT and MLRT produces the highest yields of DNA scaffolds, compared to just using one RT or the other. This required combining the two RT parts into a single plasmid.
HIV-RT and MLRT were available separately from Addgene; however, each plasmid contained illegal restriction sites. We planned to use mutagenesis techniques to remove these illegal sites, then an overhang PCR reaction of the RT genes, before finally ligating the RT parts together with a promoter, Lac(O), and terminator.

Unfortunately, our initial assembly plans were not as successful as we had hoped. Part plasmids were correctly amplified through PCR, but combining these plasmids into a single cohesive part was not successful. Several attempts were made to combine these parts, however verification via culture, DNA extraction, restriction enzyme digest, and agarose gel electrophoresis of these attempts showed that we were still unsuccessful.

Reappraoching the Design

Seeing that our first approach was failing to produce the desired results, we decided to change tactics. We realized that given the critical importance of our RT gene and the amount of time we were spending on creating it, we would be justified in spending the money to get it sequenced *de novo.* We decided upon Golden Gate assembly as the best way to put the two HIV-RT subunits and the ML-RT gene together, since we could accomplish it with a single enzyme and in a single step. We performed the assemblies once the parts arrived, ligating them into a linearized pSB1K3 plasmid with standard BioBrick restriction sites.

Verifiying the New Approach

In order to verify these assemblies, we transformed *E. coli* cells, cultured them, and extracted their DNA with a miniprep. We then conducted a restriction enzyme digest using BioBrick enzymes and analyzed the resulting fragments on an agarose gel. The bands appeared in exactly the locations we had predicted, indicating that our second attempt was a success.

![](https://static.igem.org/mediawiki/2021/e/ef/T--Virginia--images--digests_gels_%281%29.svg)

**Fig 8.** Left: The RT Full Part digested with XbaI and SpeI (expected sizes 2196bp and 5645bp). Lanes 5, 6, and 7 each have a different sample of the same RT Full Part plasmid. Right:The RT full part digested with only Xbal (expected size 7841bp). Lanes 5, 6, and 7 correspond to different samples of the RT Full Part plasmid, and are taken from the same samples, respectively, as lanes 5, 6, and 7 in Figure 2.3.

Success is not the End

From these trials and errors, we were successfully able to completely assemble the Manifold system. But our modified reverse transcriptase is only one part of the full Manifold system, which we are currently working to assemble and test.

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# Team:Virginia/Experiments

Manifold

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MANIFOLD

Synthetic Nanoreactors

[HOME](https://2021.igem.org/Team%3AVirginia)

[Main](https://2021.igem.org/Team%3AVirginia)

[PROJECT](#project)

[Description](https://2021.igem.org/Team%3AVirginia/Description)
[Experiments](https://2021.igem.org/Team%3AVirginia/Experiments)[Results](https://2021.igem.org/Team%3AVirginia/Results)
[Modeling](https://2021.igem.org/Team%3AVirginia/Model)
[Engineering](https://2021.igem.org/Team%3AVirginia/Engineering)[Notebook](https://2021.igem.org/Team%3AVirginia/Notebook)

[OUTREACH](#outreach)

[HP Introduction](https://2021.igem.org/Team%3AVirginia/Human_Practices)
[Identify the Problem](https://2021.igem.org/Team%3AVirginia/ItP)
[Imagine Solutions](https://2021.igem.org/Team%3AVirginia/IS)
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[Adjust the Project](https://2021.igem.org/Team%3AVirginia/AtP)
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[Scientific Communications](https://2021.igem.org/Team%3AVirginia/Entrepreneurship)

[TEAM](#team)

[Members](https://2021.igem.org/Team%3AVirginia/Members)
[Attributions](https://2021.igem.org/Team%3AVirginia/Attributions)

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Experiments

Index:

Experiments

Introduction

Our team has worked exceedingly hard since the start of 2021 crafting the design, assembly methods, and experimental procedures highlighted below. The project was divided into three different sections: DNA scaffold assembly, BMC assembly, and enzyme assemblies. The flowcharts under each section detail the experimental methods used to create them. By clicking on the colored boxes in the flowcharts, extra information pertaining to the assemblies and procedures are provided. There is also a key that provides additional information.

Scaffold Assembly

DNA scaffolds were produced by reverse transcription (RT) of r\_oligo genes with
HIV-RT and ML-RT. The fact that the two r\_oligo genes yield two ssDNAs sequences, which are reverse complements of each other, and can anneal, allows for the production of functional dsDNA scaffolds. To attain dsDNA scaffolds, two assemblies were performed together, as highlighted in this flowchart. The first chart is detailed to show the assembly of the  ["r\_oligo flipper"](https://benchling.com/s/seq-GpGuD42T8mtijSEKv1wx). This part is extremely useful, as it aids in the production of the r\_oligo genes. To make the r\_oligo flipper,  [Addgene plasmid GGE114](https://benchling.com/s/seq-pFokXTtV5nRUpLWoa0oq) with an empty pPSB1A3 backbone was acquired, digested, and run on a standard agarose gel. Upon its verification, sections of the plasmid were amplified by a PCR reaction, and the resulting linearized pSB1A3 backbone with BsmBI cut sites was used in a Golden Gate Assembly procedure.

For the Golden Gate Assembly, two gBlocks were inserted into the PCR product. The r\_oligo flipper remains unique because it has an HTBS sequence directly following the second BsaI site. As an effect, oligonucleotides can be easily and efficiently converted into r\_oligo genes through a Golden Gate Assembly procedure. In addition, due to the RFP insert, bacteria transformed with a successful assembly will not produce RFP, whereas bacteria not transformed successfully will produce RFP, and the colonies will be red. The addition of RFP allows for an easier and more efficient screening and detection process.

After the successful production of the r\_oligo flipper, it was utilized in addition with eight other DNA blocks in another Golden Gate Assembly, leading to the production of eight r\_oligo genes that, when reverse transcribed, code for the DNA scaffolds  [CoA 4x r\_oligo](https://benchling.com/s/seq-89HU5IClAZ5rFwIn5E2G),  [CoA 4x comp r\_oligo](https://benchling.com/s/seq-hNoWN3psq2WPiEF0CUZ6),  [ResV 4x r\_oligo](https://benchling.com/s/seq-CROxGLdAVkjuJqunISzK%20),  [ResV 4x comp r\_oligo](https://benchling.com/s/seq-N4PvzE4saE1c087JP5EA%20),  [CoA 6x r\_oligo](https://benchling.com/s/seq-UFIYz4vKvrVKp070Cq10),  [CoA 6x Comp r\_oligo](https://2021.igem.org/Team%3AVirginia/%20https%3A/benchling.com/s/seq-URGz6RwxlimWHmmcBz7o),  [ResV 2x r\_oligo](https://benchling.com/s/seq-j6UUUy0VhnLxDZCqBYoo%20),  [ResV 2x Comp r\_oligo](https://benchling.com/s/seq-pkD4HQg2PG71Y71lfALS). Some quick terminology: the “2”, “4”, or “6” in the r\_oligo genes signifies the number of copies of the pathway each scaffold will house, and “comp” refers to a plasmid coding for the complementary r\_oligo. The different r\_oligos scaffolds, ResV r\_oligos and CoA r\_oligos, were important for the proper binding and coordination of the enzymes as described in the 'Enzymes Assemblies' section.

Towards the end of this flowchart, BioBrick assembly ensured that each r\_oligo was assembled correctly with its complement in order to create self-annealing parts that produced the dsDNA scaffolds. CoA 4x and Resv 4x were biobrick assembled together, as were CoA 6x and ResV 2x. The Biobrick assemblies resulted in the last two products, which assembled different ratios of ResV and CoA scaffolds.

As depicted in the flow chart above, the actual production of the dsDNA scaffolds was made. For this to happen,  [pHIVRT\_pTp66p51](https://benchling.com/s/seq-mX1Uo1wQpdARUh3SJyyl), Human Immunodeficiency Viruses Reverse Transcriptase, and  [pMLRT](https://benchling.com/s/seq-RShdQXNQBJcSijOyfRdc), Murine Leukemia Virus Reverse Transcriptase, were assembled. Starting with a pSB1K3+ RFP empty backbone  [pSB1K3 + mRFP1](https://benchling.com/s/seq-LvCVLoSVxpdJABnd2frP%20) , a PCR procedure and purification led to its linearization with BsaI cut sites. This linearized piece of DNA was used as the backbone for a Golden Gate Assembly procedure; the HIV-RT and MLRT genes were inserted using three gBlocks, one for each subunit of HIV-RT and one for MLRT. After the Golden Gate Assembly, routine procedures were performed to acquire a diagnostic agarose gel of the plasmids, confirming RT in a pSB1K3 backbone.

For the RT in pSB1K3 to be used in conjunction with the r\_oligo assemblies on pSB1A3, the two parts had to be ligated into the same plasmid. 3A assembly moved them into a pSB1C3 backbone, and after diagnostic digests, another round of BioBricking onto pSB1A3 backbones led to the combination of RT and the r\_oligo genes (4,4, and 2,6).

BMC Assembly

Bacterial microcompartment plasmids were graciously gifted by the Warren Lab at the University of Kent in Canterbury, England, which included the “pduABJKNU” and “pduABJKNUT” sequences. Due to prior studies and modeling evidence, we opted to use the pduABJKNUT plasmid for our experiment. Its identity was verified by a supplemental sequence map through gel electrophoresis.

Two different parts of this plasmid were amplified through a PCR reaction, creating two linear DNA fragments with BsmBI sites on the ends. Once the two fragments were obtained, they were used for a Golden Gate Assembly with three PDU gBlocks and an Addgene plasmid  [pTU2-A-RFP](https://benchling.com/s/seq-SbC5q5CsnKojSVid6IMG%20), to create our composite parts,  [pduABD\*JKNUT in pTU2](https://benchling.com/s/seq-zBItmS1TiU6gbK28VbRW%20)with either CmR + TetR or just CmR,  [pduABD\*JKNUT in pTU2 Alt](https://benchling.com/s/seq-BzDRRerAUA70RQNFkaXD%20)

Enzyme Assemblies

The enzyme assemblies are detailed in the Nucleic Acids page; however, they’ll be going by numbers here. Each assembly contains different combinations of enzyme genes, and when compared to each other, the assemblies will demonstrate the greater efficiency of a full Manifold system than any of its individual components.

 [Assembly 1](https://benchling.com/s/seq-sSo6otEzYcL3hrbKNr6l)  codes for an HA Tag-ZFa-ACS fusion protein, and its assembly can be viewed by accessing the link.  [Assembly 2](https://benchling.com/s/seq-JkmzKEtDGfGunSCA1AvR) codes for a HA Tag-ZFa-ACS protein and is differentiated from assembly 1 due to the fact that the ACC beta and ACS proteins are not fusions. The link provides further insight into the design of this assembly. Assembly 3 similarly codes for a HA Tag-ZFa-ACS fusion protein, therefore it contains the same assembly as the first enzyme, however it confers TetR as opposed to CmR. Assembly 4 similarly codes for a HA Tag-ZFa-ACS protein, therefore it contains the same assembly as the second enzyme, however it confers TetR as opposed to CmR.  [Assembly 5](https://benchling.com/s/seq-6x9tPxl77tKzz2O3qoHc%20) codes for a 6xH-zif268-4Cl fusion followed by a 6xH-PBSII zinc finger-STS fusion with chloramphenicol resistance. The link can be utilized for a better visual of the assembly. Assembly 6 codes for the protein pduABJKNUT and has a pduD-ZFC fusion between pduB and pduJ and between TetR and CmR on a pTU2 ORI. In addition, everything except the CmR gene and the ORI are flanked by Tn5 transposase sites. Lastly, assembly 7 resembles assembly 6, however only contains CmR.

The enzymes were created by Golden Gate assembly reactions with BsmBI sites in addition with several other gBlocks. After the Golden Gate procedures, standard culture and plasmid extraction protocols were performed and the assemblies were confirmed by gel electrophoresis. The enzyme assemblies labeled 3, 4, 5, and 7 construct a Manifold device in which the pdu genes remain on their own plasmid. The results of these enzymes are still in the process, however be sure to check our presentation video for updates! On the other hand, the enzyme assemblies shown as 1, 2, 5, and 6 produces several versions of the Manifold device in which the pdu genes have been spliced into the genome of E. coli with Tn5 transposase. This ongoing process is still in the works, as the wet lab team is working diligently to achieve positive results.

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Manifold

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# Team:Virginia/Results

Manifold

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MANIFOLD

Synthetic Nanoreactors

[HOME](https://2021.igem.org/Team%3AVirginia)

[Main](https://2021.igem.org/Team%3AVirginia)

[PROJECT](#project)

[Description](https://2021.igem.org/Team%3AVirginia/Description)
[Experiments](https://2021.igem.org/Team%3AVirginia/Experiments)[Results](https://2021.igem.org/Team%3AVirginia/Results)
[Modeling](https://2021.igem.org/Team%3AVirginia/Model)
[Engineering](https://2021.igem.org/Team%3AVirginia/Engineering)[Notebook](https://2021.igem.org/Team%3AVirginia/Notebook)

[OUTREACH](#outreach)

[HP Introduction](https://2021.igem.org/Team%3AVirginia/Human_Practices)
[Identify the Problem](https://2021.igem.org/Team%3AVirginia/ItP)
[Imagine Solutions](https://2021.igem.org/Team%3AVirginia/IS)
[Gain Insight](https://2021.igem.org/Team%3AVirginia/GI)
[Implement the Solution](https://2021.igem.org/Team%3AVirginia/ItS)
[Adjust the Project](https://2021.igem.org/Team%3AVirginia/AtP)
[Public Engagement](https://2021.igem.org/Team%3AVirginia/Public_engagement)
[Collaborations](https://2021.igem.org/Team%3AVirginia/Collaborations)
[Contributions](https://2021.igem.org/Team%3AVirginia/Contribution)
[Scientific Communications](https://2021.igem.org/Team%3AVirginia/Entrepreneurship)

[TEAM](#team)

[Members](https://2021.igem.org/Team%3AVirginia/Members)
[Attributions](https://2021.igem.org/Team%3AVirginia/Attributions)

[AWARDS](#awards)

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[RESOURCES](#about)

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#ffd6a5

Results

Index:

Overview

Bacterial microcompartments (

BMCs![](https://upload.wikimedia.org/wikipedia/commons/thumb/2/25/Carboxysome_and_bacterial_microcompartments.jpg/800px-Carboxysome_and_bacterial_microcompartments.jpg)Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.

) were produced and extracted. The BMC shell size distribution was calculated and growth curves were determined for the cells transformed with the BMC part. Protein assays and western blot were performed to assess BMC protein concentration and identity.

The r\_oligo flipper was successfully tested via RFP colony selection and used to create DNA scaffolds containing HTBS and zinc finger binding sequences. DNA scaffolds were produced via cotransformation of r\_oligo and reverse transcriptase parts, extracted, and analyzed on agarose and polyacrylamide gels.

In order to demonstrate the functionality of the Manifold system, the BMC part, the DNA scaffold parts, and parts for various enzymes in the resveratrol biosynthetic pathway will be cotransformed in different combinations to test different aspects of the system.

Results

BMC Production

Our team was gifted the “pduABJKNU” and “pduABJKNUT” sequences for production of bacterial microcompartment (BMC) shells by the Warren Lab at the University of Kent in Canterbury, England. For our experiment, we opted to use the pduABJKNUT plasmid due to its enhanced results from prior studies and strong modeling evidence. Initially, production and extraction of both BMC plasmids was performed. BMCs were produced in order to verify that the plasmids provided by the Warren lab functioned as intended, to verify that we could successfully follow current BMC production methods for production and isolation without issues, and to obtain information regarding BMC size distribution under the culture conditions utilized. These plasmids were individually transformed into E. coli and BMC production was induced. BMCs were extracted and visualized via transmission electron microscopy (TEM). The images of extracted BMCs in Figure 1.1 and TEM images in Figure 1.2 highlight the successful extraction and visualization of the BMC plasmids, respectively.

![](https://static.igem.org/mediawiki/2021/8/8b/T--Virginia--images--vial.png)

**Fig 1.1:** Bacterial microcompartments were extracted from E. coli. The left image shows extracted pduABJKNUT BMCs and the right image shows extracted pduABJKNU BMCs. Further clarification of successful extraction was provided after the TEM images were taken.

![](https://static.igem.org/mediawiki/2021/5/53/T--Virginia--images--TEM.png)

**Fig 1.2:** TEM images of empty pdu BMCs. a.,c. pdu BMCs were composed of proteins pduABJKNU b.,d. pdu BMCs were composed of proteins pduABJKNUT. Images 4a and 4c were taken from the same sample, as were 4b and 4d. Scale bars of 0.5 μm were used in Figure 4a, 4b and 4d, while a scale bar of 1 μm was used in 4c.

According to the TEM images, the pduABJKNU BMCs were produced and isolated in a higher quantity than pduABJKNUT BMCs. In Figure 1.2c, 386 BMCs are present, resulting in a concentration of around 12.83 BMCs/μm2. In Figure 1.2d, 353 BMCs are present, resulting in a concentration of 7.40 BMCs/μm2. This observation was also qualitatively supported by the relative sizes of the protein pellets during BMC extraction (Figure 1.1). The images in Figure 1.2 were used to determine the distribution of BMC sizes in the two samples. These results are shown in Figure 1.3.

![](https://static.igem.org/mediawiki/2021/5/5c/T--Virginia--images--hist.png)

**Fig 1.3:** Distribution of empty BMC shell sizes for pduABJKNU and pduABJKNUT samples. There are 386 data points for the pduABJKNU BMCs and 353 data points for the pduABJKNUT BMCs.

In addition to the greater production of pduABJKNU BMCs, its protein concentration proved to be higher as well. The protein assays shown in Figure 1.4 were run for the two different BMCs to determine protein concentration. After the assays were completed and protein concentration was measured, the pduABJKNU Old BMCs relayed a higher protein concentration than pduABJKNUT BMCs, in which concentrations are 1.56 μg/mL and .27 μg/mL respectively. Results were run on a western blot in duplicates, and due to the difference in protein concentrations between the BMCs, only results from the pduABJKNU Old BMC appeared. The sizes of proteins are as expected for pduABKJNU BMCs. The western blot is shown in Figure 1.5.

![](https://static.igem.org/mediawiki/2021/f/fa/T--Virginia--images--proteinassay.png)

**Fig 1.4:** Protein assays of pduABJKNU and pduABJKNUT BMCs, both Old and New. The concentration of protein in each of these samples (1.56 μg/mL for pduABJKNU and .27 μg/mL for pduABJKNUT) were verified by the western blot shown in Figure 1.5.

![](https://static.igem.org/mediawiki/2021/8/83/T--Virginia--images--westblot.png)

**Fig 1.5:** Western blot run in duplicates of pduABJKNUT Old, pduABJKNU Old, pduABJKNUT New, and pduABJKNU New.

In order to obtain useful data about the growth rates of transformed BL21(DE3) cells in Ampicillin, growth curves were measured for each of the cell lines. The resulting curves are shown in Figure 1.6. Logistic regressions were then fitted to each of these curves to allow for the doubling times of the cell lines and carrying capacities in the LB solutions to be calculated. These findings are reported in Table 1.

![](https://static.igem.org/mediawiki/2021/a/ac/T--Virginia--images--growthcurve.png)

**Fig 1.6:** Growth curves for various BL21(DE3) transformants plotted against logistic regressions. The error bars assume a constant error of 5 minutes due to variation in sampling, and an error of 5% in the OD600 measurements due to observed imprecision in the spectrometer utilized. All samples were grown in LB medium containing 50 μg/mL Ampicillin except for “BL21(DE3)” (shown in blue) to which no Ampicillin was added. Plots labeled “New” were made from plates that had been stored at 4°C for one day, whereas plots labeled “Old” were made from plates that had been stored at 4°C for 6 days. The plot labeled “BL21(DE3)” was also made from a plate that had been stored at 4°C for one day.

![](https://static.igem.org/mediawiki/2021/b/b4/T--Virginia--images--stattable.png)

**Table 1:** Calculated parameters and statistical analysis of values obtained from the logistic models shown in Figure 1.6. The samples here correspond to the same cell lines in Figure 1.6. The parameters a, k, and L in the “Values” column refer to the regression parameters as shown in Equation 1. The doubling values were calculated as 60\*ln(2)/k. The carrying capacities simply refer to the value of L. And the shifts from control refer to the difference between the doubling time of sample and the doubling time of sample “BL21(DE3).” All errors were calculated according to 95% confidence intervals under a Student’s T Distribution, and error was propagated using the standard error formulas for normally distributed variables which are appropriate for use, as the t-distributions will be approximately normal under the given conditions.

![](https://static.igem.org/mediawiki/2021/3/39/T--Virginia--images--equation.png)

**Equation 1:** Three-parameter logistic equation used to compute growth curve regression models. Parameters are L, a, and k. The dependent variable is OD600 and the independent variable is t.

The growth curves shown in Figure 1.6, and the corresponding doubling values shown in Table 1 imply that there is a slight difference in the doubling time between the transformed BL21(DE3) cells in ampicillin and the untransformed BL21(DE3) cells. The transformed cells appeared to grow slightly faster on average when compared to the untransformed cells. As of now, there is no proposed mechanism for why this should be true, but it is possible that it is due to random variation in the cell lines. Additionally, there was no statistically significant difference observed between the new and old doubling times of the pduABJKNUT plasmid containing cells. Furthermore, all of the observed doubling times align with the expected doubling times for E. coli in LB medium between 20 and 30 minutes. It should also be noted that no growth was seen in the “pduABJKNU New” culture which is most likely due to the inoculation of a dead colony. The observed carrying capacities also seem to align with values found in the existing literature for most for E. coli strains when cultured in LB medium.

DNA Scaffold Production

We successfully tested the functionality of the “empty” r\_oligo flipper via RFP colony selection. In Figure 2.1, the fluorescent colonies are those with the r\_oligo flipper containing RFP instead of the sequence for a particular DNA scaffold, which in the case of Figure 2.1 is the CoA 4x part. The colonies without fluorescence are those with the scaffold sequence successfully inserted into the r\_oligo flipper via a single Golden Gate Assembly reaction. Transformed E. coli were grown on ampicillin plates as the r\_oligo flipper has a backbone containing ampicillin resistance.

![](https://static.igem.org/mediawiki/2021/4/43/T--Virginia--images--RFP.png)

**Fig 2.1:** Red E. coli colonies are those expressing the original r\_oligo flipper, which contains RFP instead of a DNA scaffold insert. Non-fluorescent colonies are those expressing the r\_oligo flipper containing a scaffold gene insert (CoA 4x) instead of RFP. Cells were grown on ampicillin plates.

The r\_oligo flipper was used to create two versions of the scaffold part via several successive Golden Gate Assembly reactions. One contains two copies of the ZF-binding domains for resveratrol enzymes and six copies of those for Malonyl CoA-recycling enzymes (Resv 2x, CoA 6x). Another contains four copies of these domains for both pathways (Resv 4x, CoA 4x). The gel in Figure 2.2 shows successful creation of both of these parts.

![](https://static.igem.org/mediawiki/2021/c/c3/T--Virginia--images--scaffold_dna.png)

**Fig 2.2:** Diagnostic digest of the (Resv 4x, CoA 4x) in lanes 2 and 3, and of the (Resv 2x, CoA 6x) part in lane 4. The expected results for all three parts are two fragments of about 1400 and 2100bp each.

An RT part was needed to allow for reverse transcription of r\_oligo genes with HIV reverse transcriptase (HIV-RT) and Murine leukemia reverse transcriptase (ML-RT) to produce DNA scaffolds. While only HIV-RT is needed to produce the scaffolds, it has been documented that the co-expression of ML-RT leads to an increase in DNA production. Thus, we assembled the HIV-RT part containing p66 and p51 subunits with the ML-RT part using a three-insert Golden Gate assembly.

We validated the success of our Golden Gate assembly through the lack of fluorescence of colonies and a diagnostic gel. We assessed the functionality of our assemblies by co-transforming E. coli with r\_oligo plasmids and reverse transcriptase plasmids, including the full parts, composites and forward sections.

![](https://static.igem.org/mediawiki/2021/9/95/T--Virginia--images--RT_part1.png)

**Fig 2.3:** The RT Full Part digested with XbaI and SpeI (expected sizes 2196bp and 5645bp). Lanes 5, 6, and 7 each have a different sample of the same RT Full Part plasmid.

![](https://static.igem.org/mediawiki/2021/1/18/T--Virginia--images--RT_part2.png)

**Fig 2.4:** The RT full part digested with only Xbal (expected size 7841bp). Lanes 5, 6, and 7 correspond to different samples of the RT Full Part plasmid, and are taken from the same samples, respectively, as lanes 5, 6, and 7 in Figure 2.3.

Co-transformation of DNA scaffold (ZF-binding domain and HTBS) parts and the full RT part containing MLRT and HIVRT in E. coli allowed production of DNA scaffolds in vivo. We grew and extracted these scaffolds via the technique described by Elbaz et al.: using an isopropyl-β-d-thiogalactoside-inducible (IPTG) promoter to form scaffolds in the presence of IPTG. DH10β E. coli cells were co-transformed with the Resv 2x, Resv 4x, CoA 4x, or CoA 6x scaffold part and RT part with each group grown with IPTG or without IPTG present. Each sample was compared with the PCR-purified dsDNA coding for its respective scaffold part. DNA scaffolds were extracted and then visualized on an agarose gel. Figure 2.5 shows the successful creation of the Resv 4x scaffold.

![](https://static.igem.org/mediawiki/2021/d/db/T--Virginia--images--scaffold_gel.png)

**Fig 2.5:** Agarose gel with dsDNA scaffolds extracted with phenol:chloroform:isoamyl. Lane 1: 20bp ladder. Lane 2: Resv 2x (not induced to make dsDNA scaffolds). Lane 3: Resv 2x (induced). Lane 4: PCR product of Resv 2x gene. Lane 5: Resv 4x (not induced to make dsDNA scaffolds). Lane 6: Resv 4x (induced). Lane 7: PCR product of Resv 4x gene. Lane 8: 1kb ladder. The ~100 bp band present in lane 6 shows successful formation of the Resv 4x scaffold.

In comparison to Genetic encoding of DNA nanostructures and their self-assembly in living bacteria by Elbaz et al

[1]Elbaz, J., Yin, P. & Voigt, C. Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nat Commun 7, 11179 (2016).

, which pioneered the r\_oligo structure, the bands on the polyacrylamide gels in the experimental groups with IPTG present show successful DNA scaffold formation and extraction (Figure 2.6).

![](https://static.igem.org/mediawiki/2021/c/cc/T--Virginia--images--scaffoldgel_ref.png)

**Fig 2.6:** Extracted DNA scaffolds from Elbaz et al.

A 3A assembly was used in order to ligate the full DNA scaffold part (2x,6x or 4x,4x and the full RT part containing MLRT and HIVRT into the same pSB1A3 plasmid. Future wetlab tasks will involve verification of this ligation via restriction enzyme digest analysis and cotransformation of this plasmid along with the full BMC part and resveratrol and malonyl CoA enzyme parts in order to test the Manifold system.

Enzyme Production

To complete the Manifold system, we needed to modify enzymes in the resveratrol pathway so that they could bind to our DNA scaffolds. This involved adding a zinc finger scaffold gene to a standard GFP protein gene. After assembly and transformation, we saw GFP presence in our cells under UV light (Figure 3.1).

![](https://static.igem.org/mediawiki/2021/2/28/T--Virginia--images--GFPzinc.jpg)

**Fig 3.1:** GFP-zinc finger fusion proteins fluorescing in E.coli.

We created resveratrol pathway enzymes with zinc finger fusions and used a Golden Gate Assembly to form the full parts. Verification of the transformed and extracted assemblies is still underway.

Demonstrating Manifold

![](https://static.igem.org/mediawiki/2021/7/7c/T--Virginia--images--1_2_3_4_5_6_7_8.svg)

**Fig 4.1** This dot chart details the final experimentation of the Manifold system. Extracted resveratrol and other measurements, if applicable, will be compared between various experimental groups to assess the functionality and benefits of a full Manifold system.

Comparison of groups 4 and 5 will determine whether differences in resveratrol production in (2x6x) and (4x4x) systems show that Manifold resolves flux imbalances in the resveratrol biochemical pathway. Comparison of groups 7 and 4 will show whether the CoA scaffold increases resveratrol yield. Comparison of groups 5 and 6 will show whether the RT part is needed to see resveratrol yield increase. Comparison of groups 2 and 3 will show whether BMCs alone do not increase resveratrol yield. Comparison of groups 5 and 8 will show whether the full Manifold system increases resveratrol yield beyond CoA and resveratrol scaffolds alone. Comparisons of groups 3 and 4, and 3 and 5 will both determine whether the full system increases resveratrol yields beyond BMCs, resveratrol enzymes and CoA enzymes alone.

As of October 21st, 2021, we are in the process of cotransforming, inducing, and gathering data on the Manifold system.

To test the affinity of our zinc finger fusion proteins to bind to our DNA scaffolds, we will run a “pseudo-southwestern blot” in which we will transfer a western blot of our scaffolds onto a membrane and wash it with purified, extracted GFP-zinc finger protein. If the DNA scaffold bands on the membrane turn green, we will know that our scaffolds can bind to our zinc finger fusion enzymes.

Finally, we also intend to do a mobility shift assay to assess the binding of our zinc finger fusion-resveratrol pathway enzymes to our DNA scaffolds.

Improving Manifold

After verification of resveratrol production, the next step in Manifold's journey, following the engineering design principle, is to test Manifold with different products. Our current experimental design presents a proof of concept by creating resveratrol, one drug of the many that Manifold could potentially produce. The next goal of Manifold is to test it at larger scales in order to determine whether it would be useful in the chemical industry. We hope to accomplish this by efficiently engineering Manifold such that the least amount of input and costs yield the greatest amount of product. Streamlining this process will help us expand to a larger-scale manufacturing of drugs and chemicals as needed by the industry.

Another key way for us to improve Manifold is to continue modeling its molecular dynamics in order to better understand the specifics and possible restraints of the system. This comes through understanding how the BMCs are being created, the assembly of our scaffolds, and the attaching of enzymes. Using modeling as a tool to visualize this data, we hope to improve our scaffold and enzyme design so that Manifold can truly be a platform technology.

Another future goal of Manifold focuses on outreach through presenting our system to other companies and labs as a technology that can be utilized to improve their own drug manufacturing.

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