# CAGEN draft

The Critical Assessment for Genetically Engineered Networks (CAGEN, pronounced "cajun") is a new competition designed to improve the robustness and performance of human-designed biological circuits and devices operating in cells. The competition is intended to bring together leading research groups in biological circuit design to compete to demonstrate their abilities at designing circuits that perform in a prescribed manner in a variety of cellular contexts. Each year, a steering committee will select one or more challenge problems that involves the design of an increasingly complex set of biological functions in a range of environments. Teams must submit their sequences, plasmid DNA implementing their circuit and data characterizing the performance of their system against a specified test suite. The top 3-5 designs will be submitted to the NSF BIOFAB for final characterization, and the winner will be selected based on a set of quantifiable metrics.

### CAGEN Challenge Guidelines

- Challenges should specify tasks that, if achieved, would imply that significant improvements in the state of the art have been made. The descriptions should as much as possible be agnostic as to the approach: circuit design, organism used, etc. should not be specified. On the other hand, it should be clear from the stated metrics that the task is not doable without a robust design.
- Challenge descriptions will be posted on the CAGEN website along with discussion threads for at least one month. The descriptions can be be revised during this time.
- The author/moderator can request that the challenge be approved by the CAGEN board, after which time it is posted as an official challenge.
- Changes to the challenge description can be made yearly, after the awards (if any) for that year are made.
- Contestants submit publishable work to CAGEN along with a description of how the work addresses the challenge. There will be a yearly due-date.
- The CAGEN board assigns reviewers for each challenge. The reviewers use the metrics defined in the challenge description to evaluate each proposal. The following outcomes are possible:
- No award: none of the submissions significantly improve the state of the art.
- A single award: one of the submissions meets the metrics in the challenge and substantially improves the state of the art.
- A tie: multiple submissions offer a similar level of improvement on the state of the art.

# CAGEN Challenge Proposals: 2011-12

The following challenges have been proposed as possible CAGEN challenges for 2011-12.

## Robust Gene Response

**Synopsis:** The goal of this challenge is to design a circuit that can express a fluorescent protein at a controlled level upon the introduction of a chemical inducer, with minimal variation in expression between cells and in multiple contexts. At conditions yielding maximum expression, the circuit should quickly bring the volume-normalized fluorescence from 1X to 10X in response to the addition of an inducer of the designer's choice. The circuit must work at multiple temperatures, with minimal variation in the fluorescence over time, operating temperature and cell choice.

**Motivation:** Current synthetic circuits demonstrate large variability in expression level when operating different contexts and this limits the ability of synthetic biologists to build on designs performed by other groups. By designing circuits that demonstrate highly repeatable performance over a range of operating conditions, it will be possible to make better use of designs in a modular fashion.

**Impact:** Improved understanding engineering processes for synthetic biologists will enable more rapid and pervasive development of synthetic circuits, with applications in materials processing, environmental science, agriculture and and medicine.

**Metric(s):** The winner of this challenge will be determined based on the worst case, mean square error between the ideal step response and the experimental results, with evaluation over multiple temperatures. To be considered, data for the circuit must be submitted for steady state operating temperatures at a nominal value (chosen by the contestant), nominal + 5% and nominal - 5%, with measurements taken in at least 5 individual cells chosen from separate colonies. This represents a set of 15 total time traces of data. At least one of these responses must demonstrate a step response that goes from 1X to 10X expression level in response to the addition of the inducer.

The following method will be used to determine the numerical score for each time response: let <amsmath>r_i(t)</amsmath> represents the (single) ideal step response at a given induction level i (with minimum and maximum values chosen by the participant), <amsmath>y_i(t)</amsmath> represents the measured fluorescence of a given cell, T1 represents the time at y(t) reaches 5% of its maximum value and T2 represents that point at which it reaches 95%. Each run will be scored according to the formula:

\text{Score[run]} = \int_{T1}^{T2} |y_i(t) - r_i(t)|^2 dt</amsmath>

The score for the submitted design will be the worst (highest) value of the score across all runs (15 total). Note that <amsmath>r_i(t)</amsmath> is fixed based on induction level, while <amsmath>y_i(t)</amsmath> depends on the specific run. The participant can specify a single minimum value for <amsmath>r_i(t)</amsmath> and a table of maximum values (one for each induction level).

## Universal Logic Gates

**Synopsis:** This challenge requires the demonstration of a scalable and robust NAND (“not and”) or NOR (“not or”) logic gate. Successful designs will allow the use of multiple coupled gates in a cell to compute arbitrarily complex logic. Circuits should employ commonly available inputs (e.g. IPTG) and output a fluorescent signal.

**Motivation:** Every Boolean logic gate can be constructed entirely out of NAND gates, and can also be constructed entirely out of NOR gates. For this reason, NAND and NOR gates are said to be universal‹they can be wired together to express arbitrarily complex logic. The main difference between electronic logic gates and current implementations of biological logic gates is that current biological logic gates cannot be used in multiple contexts in a single cell. Constructing complex logic statements out of universal gates in biological systems will require a rethinking of current biological logic gate design.

**Impact:** The availability of truly scalable logic gates will enable the construction of multiple complex circuits which can be linked together in a modular fashion. This would provide a basic framework for the the construction of complex synthetic devices such as biological counters and sensors which can give a defined output based on a combination of complex inputs. Applications would be diverse, including basic biology, medicine, environmental science, and agriculture.

**Metric:** Competitors should demonstrate the construction of three Boolean logic gates using various combinations of a single universal logic gate. Entrants will be judged based on:

- Switch-like response of device to inputs
- Complexity with regards to the logic statements constructed (i.e. an XNOR gate is more difficult to construct with NAND logic than an OR gate)
- Parsimony with regards to the number of gates in the circuit (e.g. an optimal OR gate will be made out of 3 NAND gates)

Selection of the winner shall be done by a jury consisting of the CAGEN steering committee.

## Robust Synthetic Development

**Synopsis:** The goal of this challenge is to design a circuit that can express a fluorescent protein with specific self‐generated spatial patterns. These patterns should ideally emerge without external spatial cues. The two basic patterns we consider are: (1) A circular pattern with a diameter of exactly N cells, where cell are on at the center of the circle and off outside (2) A striped on‐off pattern with a wavelength of exactly N cell diameters. The boundaries formed should exhibit a sharp turn‐on and turn‐off functions on the order of one cell length with minimal errors (no off cells in the on area and vice versa).

Synthetic developmental patterning challenge. The ideal circular (left) and striped (right) patterns for this challenge. Boundaries for challenge #2 may be determined by external restriction of growth region. |

**Motivation:** Motivation for this challenge is twofold: (1) Current synthetic circuits are limited in their ability to form self emerging spatial patterns in a robust manner. In fact, most current synthetic patterning circuits are driven by external cues. This challenge will likely push forward the technology for developing synthetic circuits based on cell‐cell communication (2) By constructing synthetic circuits exhibiting robust self emerging spatial patterns we can learn about the mechanisms involved in developmental patterning circuits. In particular, such a challenge would help explore the range of possible genetic circuits that can generate multicellular development and their limitations.

**Impact:** The impact of this challenge is twofold: (1) Improved understanding of basic engineering principles for synthetic biologists will enable more rapid and pervasive development of synthetic circuits, with applications in materials processing, environmental science, agriculture and medicine. (2) The ability to generate self emerging spatial patterns may be important in the field of tissue engineering where precise and accurate patterns of differentiation are required.

**Metric(s):** The winner of this challenge will be determined based on the difference between the observed mean spatial response function and an ideal spatial pattern. The ideal pattern for the concentric ring challenge ( #1) is on at a diameter of N (TBD) cells and turns off sharply at beyond that diameter. The ideal pattern for striped pattern challenge (#2) is an on and off patterns with a fixed wavelength of N (TBD) cells. Again, boundaries between on and off states should be sharp.

- The following method will be used to determine the numerical score for each submission: let <amsmath>r_i(x)</amsmath> represent the ideal spatial response curve (for the concentric ring: Fmax for R<RN, 0 for R>RN, where RN represent the diameter of N cells, and Fmax is a maximal fluorescence level (determined by contestant)). Let <amsmath>y_i(x)</amsmath> represent the measured fluorescence of a given cell at steady state (time will be determined by contestant). Each run will be scored according to the formula:

\text{ Score[run]} = \int_0^\infty |y_i(x) - r_i(x)|^2 dx</amsmath>

A similar scoring method will be applied to the striped pattern.

The score for the submitted design will be the worst (highest) value of the score across 3 runs each at 3 temperatures: nominal, nominal-5%, nominal+5% (where nominal is chosen by the contestant). Final determination of the winner will be done by a jury consisting of the CAGEN steering committee.

## Competitive Gene Expression

**Synopsis:** This challenge is to engineer a cell that maximizes production of a reporter molecule, while competing for resources with a cell lacking the engineered circuit. Equally-sized inoccula of the engineered strain and the wild-type strain are added to a batch culture and grown to saturation. The winning team has the highest reporter yield.

**Motivation:** Synthetic biosynthesis of protein and small molecule targets is often limited by genetic instability of engineered strains and competition for resources with non-producing mutants. This is particularly true for valuable but cytotoxic molecules (for example, antibiotics such as carbapenems). Costly fermentation processes might become more reliable if we could engineer strains that i) make more efficient use of resources, or ii) can actively inhibit accumulation of non-producers.

**Impact:** This challenge could impact the practice of industrial biotechnology by providing new tools and strategies for maximizing heterologous product yield in batch fermentation. Indirectly, development of these tools would be useful to the broader community of biological engineers interested in genetic stability, control of diverse populations, and circuit design.

**Metric(s):** The product will be fixed for a given edition of the challenge; for the 2011-2012 CAGEN challenge, the reporter molecule will be GFP. (In subsequent editions, the product could have greater significance or level of public recognition [e.g. a flu drug].) Product yield across three replicate batch flasks will be measured for each group, normalized to some limiting nutrient. The overall winner will be determined by a jury consisting of the CAGEN steering committee, based on the highest yield. The jury may award additional prizes for novel mechanisms.