Presented by Team BostonU 2012

Monique De Freitas¹, Shawn Jin¹, Evan Appleton², Swati Carr², Sonya Iverson², Traci Haddock³, and Douglas Densmore^§

¹iGEM Student Team Member, ²iGEM Team Mentor, ³iGEM Team Primary PI, ^§Faculty Sponsor, Department of Electrical and Computer Engineering, Boston University, Boston, MA, USA

Abstract

Our project has three aims: to introduce MoClo as an alternative assembly technique to BioBricks, to develop a standardized protocol for the characterization of genetic circuits using flow cytometry, and to share our MoClo Kit with the iGEM community. MoClo is an assembly technique developed by Weber et al., 2011, which involves a multi-way, one-pot digestion-ligation reaction, enabling faster and more efficient construction of genetic circuits. We converted a large subset of BioBricks from the Registry into MoClo Parts using PCR and cloning strategies. We are in the process of building and characterizing various genetic circuits using MoClo Parts, which we will compare to their BioBrick counterparts. A characterization workflow will be shared once this is complete. We also developed a data sheet using Clotho to be included in the Registry of Standard Biological Parts for each Part we characterized to easily share our data with the synthetic biology community.

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Project Goals

To introduce MoClo as an alternative assembly method for use by iGEM teams and start a library of MoClo parts for future iGEM teams to use

To develop a characterization workflow for genetic circuits containing fluorescent proteins and share this protocol with the iGEM community

To generate a data sheet that contains information on the device, parts, assembly technique(s), and characterization

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Motivation

We identified 3 areas of synthetic biology that we wanted to improve for the iGEM community: Building, Characterizing, and Sharing.

BUILDING: BioBricks assembly requires multiple cycles of ligations and digestions, making it very time consuming when constructing large circuits

CHARACTERIZING: Characterization methods for circuits containing fluorescent proteins vary, making data comparison challenging

SHARING: Parts pages on the Registry often lack a standard format in which characterization information is displayed

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Building: MoClo Assembly Method

Modular Cloning, or MoClo, is a relatively new assembly method based on Golden Gate introduced in 2011 by Ernst Weber et al., which uses Type IIS restriction enzymes (we used BpiI and BsaI) to generate 4bp overhangs. These 4bp overhangs are called fusion sites, which must match between parts in order to ligate them together.

This allows the user to ligate up to six DNA parts together in a one-pot reaction, cutting down the time it takes to build large circuits dramatically (shown below).

There are three Levels of MoClo Parts (shown below):

Level 0: Basic part (ex: promoter, RBS, CDS, etc.)
Level 1: Transcriptional unit (up to 6 Level 0 Parts)
Level 2: Composite of up to 6 Level 1 parts

Reference: Weber et al., PLoS One 2011

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Building: MoClo Library

We converted 31 BioBricks and 4 new parts into Level 0 MoClo parts to create a library of MoClo parts.

For BioBrick parts >100bp, we used a standard PCR methodology.

We used a ligation PCR methodology for sequences <100bp in length (J. Lee et al., Biotechniques. 2004).

All parts listed in Table 1 were confirmed with sequencing

All of our parts and primers are stored in a Clotho database (Xia et al., Methods in Enzymology 2011).

New Parts
We have cloned a new copper sensitive σ⁵⁴-regulatory system: pMmoR is induced by mmoR, which is repressed by copper (J. Scanlan et al., 2009).

Two new fluorescent proteins, EBFP2 and iRFP, have also been cloned.

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Characterizing: Workflow

We have begun developing a flow cytometry workflow for the characterization of genetic circuits that contain fluorescent proteins in E. coli (shown below).

Parameters that often vary between labs is the time of growth, culture shaking speed, E. coli strain used, and media used. Other important parameters include the lasers, filters, and settings used in the flow cytometer for measurement.

We are in the process of testing these variables to determine the parameters that will work best for the genetic circuits we have built using BioBricks.

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Characterizing: Inverter

We characterized an inverter built with BioBrick parts (shown below) using the parameters given in previous Characterizing box.

This first pass at developing a flow cytometry workflow showed promising results, with the GFP increasing and RFP decreasing smoothly as the arabinose concentration increased.

This will be expanded to include our MoClo Level 1 and Level 2 parts once built. We plan to recreate this inverter using MoClo.

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Sharing: MoClo Kit

We have submitted all of our Level 0 MoClo parts and cloning vectors for Level 0, Level 1 and Level 2 to the Registry as part of our MoClo Kit.

It is our hope that future iGEM teams will contribute to this library of Level 0 MoClo parts so iGEM can move away from binary assembly and towards this modular, one-pot assembly technique.

This figure shows the number of DNA parts in our MoClo Kit and the power of MoClo as an assembly technique.

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Sharing: Datasheet

We have also begun work on generating a data sheet with the following categories of information represented:

General Information (Author, date, Brief abstract, links to relevant sources)

Part Information (Device design and composition, Assembly method(s))

Growth/Measurement Conditions (Specific experimental conditions, List of assays used)

Data Analysis (Gel images, Sequencing data, Flow cytometry analysis, Other assay analysis)

We plan to pull this information from our Clotho database so any Clotho user can generate these data sheets for their parts.

A prototype data sheet for our submitted part (BBa_K783067).

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Current and Future Work

We are building more Level 0 parts and will submit them to the Registry when confirmed

We will make fusion proteins to combine functional genes and fluorescent reporters using MoClo

We are modifying the growth parameters for our characterization experiments to include 96-well plates

We will test our parameters on multiple flow cytometers

We are writing the RFC standard for MoClo and will submit that to the BioBricks Foundation within the next month

We will complete and share the code to make our data sheet so future iGEM teams that use Clotho can easily pull together all of the information required for their Registry pages

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Contributions to iGEM

BUILDING:Converted 29 BioBrick parts into 31 MoClo Parts (Foundation for converting iGEM to MoClo) Contributed 4 new parts, for a total of 35 parts

CHARACTERIZING: Characterization workflow for circuits with fluorescent proteins in development with shown success

SHARING: Shared our MoClo Kit for future iGEM teams to use and outlined a data sheet to implement for Clotho users

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Acknowledgements and Sponsors

Acknowledgements
Dr. Swapnil Bhatia
Janoo Fernandes
Dr. Michael Smanski
Dr. Ernst Oberortner
Jenhan Tao
Dr. Alexy Vorobev

Sponsors

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