Synthetic Biology
Support and Educational Content

Measuring promoter-driven transcriptional noise in E. coli

Team William & Mary use IDT gBlocks® Gene Fragments and custom oligos to characterize expression stochasticity of popular iGEM promoters


William and Mary

Image courtesy of iGEM.

With 8 members and only one previous iGEM appearance, the College of William & Mary’s undergraduate team was relatively understaffed and inexperienced at the 2015 Giant Jamboree. In fact, many of the 250+ teams in the competition had never heard of the Virginia-based school, and when less than 50 people attended their initial presentation the team certainly felt like underdogs. Yet as the 5-day competition in Boston came to a close, Team William & Mary walked away with 4 first-place honors, including the Grand Prize.

The 2015 iGEM team, led by senior Andy Halleran and junior Caroline Golino, was an impressive interdisciplinary mix of students, whose range of study included biology, chemistry, mathematics, computer science, and neuroscience. Once selected, the 8 students began brainstorming project ideas, which is not always easy for such a diverse group. However after weeks of reading literature and crossing ideas off the chalkboard, the group agreed on a topic that was not only relevant, but also took advantage of their interdisciplinary skillsets.

Characterizing stochasticity levels for popular iGEM promoters

Stochasticity, otherwise known as noise, can be thought of as the amount of variation that occurs within a system. In dynamic biological processes such as gene expression, noise is inevitable; however, it is common for some transcriptional processes to be more “noisy” than others. For their iGEM project, the William & Mary team characterized promoter-driven transcriptional noise in E. coli. Halleran explained, “There has been a surge in variability studies involving natural gene expression in eukaryotes, but less is known about expression variability as it pertains to synthetic biology. Specifically, very little had been published about the level of variation that exists for the most commonly used promoters in synthetic biology. We thought this should be very important information for anyone building gene circuits and gene networks that actually work.”

To determine the noise level of common promoters, the students expanded on research done by Michael Elowitz and colleagues in 2002. Elowitz et al. defined noise as having both intrinsic and extrinsic components, and he evaluated these using a dual-reporter fluorescent system [1,2]. In this model, 2 constructs are created with identical promoters, which each drives the expression of a distinct fluorescent protein—one construct includes Yellow Fluorescent Protein (YFP) and the other uses Cyan Fluorescent Protein (CFP). Researchers can use this system to differentiate between a promoter’s extrinsic factors, which should affect both constructs equally, and intrinsic factors, which lead to a difference in the ratio of YFP:CFP expression.

The William & Mary iGEM team applied Elowitz’s promoter evaluation method to the 3 most frequently used promoters in the iGEM BioBrick registry—the collection of genetic parts used by iGEM teams to build synthetic biology systems. These are the lacI-repressed promoter (pLac), the lacI-repressed lambda pL hybrid promoter (PLlac01 Hybrid), and the lambda cI-repressed promoter (cI repressible). To accurately measure promoter noise, the students needed to ensure that cells only contained one copy of each promoter. To control this, they integrated their dual-fluorescent constructs into the E. coli genome using gBlocks® Gene Fragments and the Gibson Assembly® method. They also fused an antibiotic to the construct, so that they could select for the cells that underwent successful integration. The resulting noise levels for the 3 promoters are shown in Figure 1.

Noise measurements during gene expression

Figure 1. Common promoters have varying levels of noise during gene expression. Intrinsic, extrinsic, and total noise levels were measured for 3 promoters in E. coli, where R0010 = pLac, R0011 = PLlac01 Hybrid, R0051 = cI repressible. Each promoter was measured once (sample size n = 1). Y-axis, noise (n2) = σ2 / μ2.

The BioBrick registry characterizes these promoters as if they all function similarly; however, the team’s data suggested otherwise. Their project demonstrated that promoter R0011, the lacI-repressed lambda pL Hybrid promoter, possesses significantly less intrinsic noise than the others. This finding was not only encouraging for the William & Mary students, but it will also help iGEM teams select promoters for their projects in the future.

A modified Cas9 for CRISPR-based promoter inhibition

In addition to characterizing noise variability in common promoters, the team also introduced new tools for manipulating promoter expression using a CRISPR/Cas9 approach. Due to new advances in Cas9 technology, one can now remove Cas9 catalytic nuclease activity while keeping DNA-binding properties intact. Such a Cas9 variant, known as dCas9, has led to the emergence of novel methods for controlling gene expression [3]. The William & Mary students built a codon-optimized dCas9 that, when used with an appropriate guide RNA (gRNA), reduced observed gene expression by 97% in E. coli. In this “side project”, which the team admits was a substantial amount of work, they created 12 gRNAs that targeted promoters commonly used for iGEM. Halleran notes, “These experiments really meshed with the whole idea of our project. Going forward, if we want to build larger, more complex, more sophisticated regulatory networks, we don’t just need information about underlying variabilities. We also need the tools to implement these networks. With the parts we built, someone can now repress a promoter that would normally not be repressible.”

IDT: A proud supporter of William & Mary’s innovative achievements

IDT’s special offer of 20 kb of free gBlocks® Gene Fragments (available to all iGEM teams), along with numerous custom primers, proved invaluable to the William & Mary team’s success at iGEM. They used the offer in its entirety and noted that the dCas9 side project would not have been possible without it. The group is extremely proud of their work, and they should be. The 2015 Giant Jamboree showcased some of the most sophisticated synthetic biology projects in the world. But few teams were as thorough, thoughtful, and impactful as the William & Mary team. As iGEM judge Markus Gershater says, “This was a piece of work that will likely be a foundation of how iGEM teams characterize and use these parts in the future, leading to much more robust and reliable biological designs [4]."

In addition to winning the Grand Prize, the team received first-place honors for Best Measurement Project, Best Education and Public Engagement, and Best Presentation. Read more about their project at http://2015.igem.org/Team:William_and_Mary.


References

  1. Elowitz MB, Levine AJ, et al. (2002) Stochastic Gene Expression in a Single Cell. Science, 297:1183–1186.
  2. Swain PS, Elowitz MB, Siggia, ED. (2002) Intrinsic and extrinsic contributions to stochasticity in gene expression. PNAS, 99:12795–12800.
  3. Qi, LS, Larson MH, et al. (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5):1173–1183.
  4. McClain, Joseph. (2015) iGEM Grand Prize: Try to grasp ‘the magnitude of what they have achieved’. The College of William & Mary News & Media. (Accessed January 25, 2016.)

Product focus—custom dsDNA, oligos and primers, design tools


Custom dsDNA fragments

gBlocks® Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–2000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications, including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.


Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.


Custom oligonucleotides and primers

You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides/primers, and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on. Learn more or order now.


Free online tools for oligonucleotide analysis and primer design

Explore IDT's free, online software for oligonucleotide analysis and for qPCR probe and assay design. The design engines for these tools use sophisticated algorithms that take into account factors such as nearest-neighbor analysis and buffer conditions to calculate Tm.


Alt-R CRISPR-Cas9 System

Increase efficiency of genome editing using the Alt-R CRISPR-Cas9 System, which includes all of the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods.

Related reading

iGEM teams engineer success with gBlocks® Gene Fragments—By August, 2015, this year’s iGEM teams had made significant progress on their projects. We spoke with some of the teams to find out how the competition was going and how they were using gBlocks Gene Fragments.

iGEM students engineer biological tools for a better world—Projects from 2 of the prize-winning 2013 iGEM teams show how non-standard natural and synthetic amino acids can be used in 1) peptide synthesis, and 2) tuberculosis monitoring and treatment. Both projects make use of gBlocks Gene Fragments to speed up construct assembly.

The gene construction revolution—See how use of high-quality, custom dsDNA fragments as a starting material allows you to turn what might otherwise be multi-step cloning assemblies into simpler reactions. You can often just order an entire target sequence that is ready for cloning or other uses.

Getting started with Alt-R CRISPR-Cas9 genome editing—Learn about the components of the Alt‑R CRISPR-Cas9 System, get information on designing Alt-R CRISPR crRNA oligos, and review the genome editing protocol from the user guide.

Author: Nolan Speicher is a scientific writer at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.



gBlocks® Gene Fragments

Double-stranded DNA up to 3,000 kb—great for easy gene construction, CRISPR genome editing, and more.

Learn how ≫


Related Articles

CRISPR and Cas9 for Flexible Genome Editing

Applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) for targeted gene editing or removal of sequences.

Read more ≫

Cloning Strategies Part 3: Blunt-End Cloning

Tips and tricks for blunt-end cloning.

Read more ≫

Building Biological Factories for Renewable and Sustainable Products

Amyris uses genetic engineering and screening technologies to design microorganisms that convert plant-sourced sugars into target molecules, including pharmaceuticals, biofuels, polymers, flavors, and fragrances.

Read more ≫

iGEM Students Engineer Biological Tools for a Better World

iGEM teams show how non-standard natural and synthetic amino acids can be used in 1) peptide synthesis, and 2) disease monitoring and treatment. Both projects make use of gBlocks® Gene Fragments to speed construct assembly.

Read more ≫

Creating a Synthetic Immune System for Optimized Immune Profiling

A high-throughput method for sequencing and quantification of rearranged antigen receptors on T- and B-cells using gBlocks Gene Fragments to create a “gold standard synthetic immune system.”

Read more ≫