Endy:F2620/Nature Biotech draft

First Prelim Draft

(28th March 2006) This is first preliminary draft. I was wondering whether it is going in the right direction? [1]

D1 Corrected by Barry

(30/03/06)

Title

Characterization of BBa_F2620, an engineered cell-cell communication device

Authors

Anna Labno[1], Barry Canton[2], Drew Endy[2]
1. MIT Biology & Physics
2. MIT Biological Engineering

Abstract (3 sentences, 70 words)

The rational design and construction of synthetic biological systems will be enabled by the ability to predict the behavior of a system from the characteristics of the devices that comprise it. I feel this change of words makes it sound better but change of structure makes it sound like a definite thing that it is going to happen, while the previous one was more like: we are making it happen. (ania) We engineered and systematically characterized a cell-cell communication device, BBa_F2620 by measuring the transfer function (including switch point, latency, and variation), the input signal specificity, and the device stability. These characteristics are specified on the device datasheet presented here. Families of devices characterized in the manner described here, will accelerate the development of larger-scale systems.

Body

What is the problem

Current research in synthetic biology aims to enable the design and construction of biological systems in the manner that we now engineer electrical, mechanical, and other man-made systems. Biological systems offer properties and functions not found in traditional engineering systems and have broad applications in medicine, biology and engineering. To enable the design of multi-device systems, it is necessary to be able to predict system function from the constituent devices of the system. can we maybe use word "system" less?, is there a synonym? (ania)Such prediction of system function requires that the behavior of the constituent devices be ((this may be ESL, but why not "is"?)) well specified and documented. Furthermore, this characterization of devices must be done in a consistent manner such that characterized devices can be composed reliably. While a wide range of devices and systems have been described (refs) there has been insufficient characterization of those devices and systems to allow the rapid, reliable assembly of more complex systems.

What is our solution to the problem

We describe a set of characteristics that specify the performance of a simple biological device. Furthermore, we have measured these characteristics for a cell-cell signaling reciever device, F2620. A fluorescent reporter device was used to measure the output of the receiver device. by applying a similar approach to other devices, a library of well-characterized and composable devices could be generated.

Background on F2620

Cell-cell communication allows individual cells to coordinate their behavior with the rest of the population and as such is a powerful technology for engineering complex biological systems. F2620 is a receiver device that responds to the concentration of a small signaling molecule (an acyl-homoserine lactone or AHL molecule) in the extracellular media by modulating the transcription rate from a promoter. Hence, we define the input to the device to be extracellular concentration of AHL and the output to be transcription rate. The device is based on elements of the quorum sensing system of Vibrio fischeri. The quorum sensing system includes an enzyme, LuxI, that synthesizes an AHL molecule (N-(b-ketocaproyl)homoserine Lactone). LuxR is a transcriptional activator protein that is active when bound to AHL. When active, it binds to the Lux box and recruits RNA polymerase to the operator region. F2620 contains 5 standard parts. The first 4 parts constitutively express LuxR as shown in Figure X. The fifth part is a promoter that contains a LuxR binding site. This promoter is the rightmost part of the V. fischeri Lux operator. To measure the output from F2620 we connected a GFP reporter device, E0240 downstream of F2620. I think this part may be challenging for people who aren't in the field to understand. First of all there are six "pieces" in the picture and it is not immediately obvious that we consider two terminators to be a "one part". Also when you say "first 4 parts express" it seems to me like they are all protein generatators. Maybe "are involved in expression". I don't know about Nature, but maybe for J&J we should step people slower through this. What do you think about: "F2620 consist of six standard parts (Figure 1). TetR promoter (R0040), followed by ribosome binding site (B0034), drives expression of luxR from LuxR protein generator (C0062). Transcription from TetR promoter is terminated by two transcription terminators (B0010, B0012) to ensure full termination. The sixth part is a LuxpR promoter (R0062) that contains a LuxR binding site. This promoter is the rightmost part of the V. fischeri Lux operator. To measure the output from F2620 we connected a GFP reporter device, E0240 downstream of F2620 (not shown on Fig 1)."

Choice of relevant device characteristics; Choice of methods for characterization;

The transfer function relating device input to output is the primary characteristic for any device. For the receiver device, we calculated the input by adding a known concentration of AHL to the culture media. We measured the output by calculating the rate of GFP accumulation per colony forming unit (cfu). We derived certain parameters that capture the key characteristics of the transfer curve - Hi/Lo input and output values, switch point, performance variability between genetically identical clones, input signal specificity, latency, and device stability (genetic and performance). The GFP reporter device was chosen because it allowed reliable, high time-resolution measurements to be made via multiwell fluorimetry and flow cytometry.

Brief narrative on experimental work; Comments on specific experimental results;

[likely given one page article, we should embed discussion points with results directly]

The maximum output level, Hi value, was determined to be ### GFP/cfu.s and was observed above an input of 10E-7M AHL. The device was considered to be off (Lo value) when GFP accumulation rate was below 10% of the maximum output, which occurred below 10E-9M AHL. The switch point for the device, the input concentration at which output is at 50% of the maximum is 10E-9M AHL. (Why is this the same as the lo input? - because I made a mistake -corrected in the new version (ania))

We measured the performance variation between genetically identical colonies taken from long-term storage using multiwell fluorimetry. The average performance of cultures grown from 6 colonies is #### GFP/cfu.s. The coefficient of variation in the Hi value among the 6 colonies is ##% and is evenly distributed above and below the mean. Other tested concentrations, show much lower variation, with coefficient of variation below ##% (see Fig. X).

We sought to quantify the ability of the device to distinguish between its cognate inducer AHL (N-(β-Ketocaproyl)-DL-homoserine lactone) and a range of chemically similar inducers with varied length side chains (list here). Fig. X shows transfer curves obtained using the different AHL molecules as inputs. The maximal output of the device (Hi level) shows strong dependence on the specific inducer. The cognate AHL produces the highest output level of ### GFP/cfu.s. A similar inducer lacking a carbonyl group and having chain length intact or extended to 7, 8 or 10 carbon atoms show response decreased by less then ##% with respect to maximum output. When the AHL molecules have their side chains extended further to 12 or 14 carbon atoms or shortened to 4 carbon atoms, activation is visible, but its maximum level is less than ##% of the cognate inducer at maximum output. It can be seen that the switch point for each of AHL variants is constant at 10E-9M.

Latency is defined as the time delay between a change in input concentration and the output level reaching 95% of its final value. These values were obtained by measuring the rate of GFP accumulation per cfu at a high induction level every minute in a multiwell fluorimeter until a constant accumulation rate was obtained (data not shown?). The rate reaches a plateau of #### GFP/cfu.s after 12min. Not yet quite sure what we should say about on to off. Technically, on to off involves washing the AHL out of the media-BC. and then after the transcription is stopped using Rifampicin the device output decreases to reach Lo value after 40min. This implies on/off latency of 12min shouldn't it be less than this if 12mins is the time to steady state?-BC and off/on latency of 40 min for the receiver-reporter construct.

Device stability was investigated under different operating conditions by propagating the culture through 94? doublings over the coure of 5 days. Performance under low input conditions, assayed using multi well fluorimetry, shows slight variations in GFP accumulation rate over the course of the experiment (coefficient of variation ###%). The performance of the device working under high input conditions shows similar variations during first three days of the experiment; however, in the fourth day the high output level dropped to approximately ##% of the original level and on the fifth day the high output had fallen further to ## (data not shown?). In order to gain more insight into the mechanism of failure, single-cell performance was investigated using flow cytometry and showed that the population of cells split on day 3 (how many doublings) into two groups: a more populous one, which was not-activated and a less populated one (quantify relative size of populations), which still retained fluorescence (Figure 1). On the last day there few visibly fluorescent cells. The DNA sequence remained unchanged over the course of the experiment when the device was operated with low input. When operated with a high input, a fraction of the cells acquired a mutation in the receiver sequence that ###

Comments on specific experimental results

[likely given one page article, we should embed discussion points with results directly]

Significance and Future directions

This work presents a first attempt of comprehensive characterization of a standard biological part, which has a multi-fold importance. In the process of characterizing BBa_F2620 we lay out the basis of an engineering methodology for the future characterization of biological parts and populated a first-generation datasheet that describes the use and operation of BBa_F2620. We hope that the biological engineering community will begin to work together to populate a library of well-characterized devices in a manner similar to that described here, to facilitate engineering of complex biological systems.

SupMat

(note, we can develop this stuff here but everything should end up on the RoSBP)