Technical note: LFNS-SMOD-0.7

smod Smart Measuring Optical Device

 

™  

Recombinant protein expression in bacterial cultures using the SMOD

Abstract

The SMODTM or Smart Measuring Optical Device, is a sensor that monitors cell growth

in situ (OD600) and transfers data in real-time

to a Windows PC. The device and data are

managed by SMOD Control Software (v1.0).

In this technical note we demonstrate how

the SMOD can be used to obtain the optimum induction time for protein

expression and monitor the culture during

protein expression.

Authors Grand, R. S., Seymour, C., Lifeonics Ltd.

Keywords Protein expression, optimized induction,

Arabinose, IPTG, OD600 and temperature measurements.

Copyright © 2016, Lifeonics Ltd.

Introduction

The expression of recombinant proteins in bacterial cells is a common biological technique used

to produce large amounts of soluble protein for basic research and biomedical applications.

However, the optimization of protein expression to obtain maximum protein yield is a laborious

task that includes testing various Optical Densities (ODs) for induction, concentration of the inducer (e.g. Arabinose, IPTG) and incubation temperature. The Smart Measuring Optical Device

(SMOD) simplifies this task by providing automated, real-time measurements of OD and

temperature in situ that can be monitored from any computer device.

Benefits of the SMOD

The SMOD is a biophotometer that is placed into the cell culture at the beginning of an

experiment. Together with the SMOD Control Software, the SMOD enables the user to measure

OD600 and temperature at time intervals of choice (as frequent as every 10 minutes) and relays the data in real-time via Bluetooth to a control computer. The data can then be visualized on any

device (e.g. Computer, Tablet, Smart-phone), providing accurate data to enable informed

decision making and freeing up users time for other tasks.

How the SMOD can help for protein expression

First, the expression of recombinant proteins needs to be optimized, which includes testing

various ODs for induction of protein expression, different concentrations of the inducer (e.g.

Arabinose, IPTG) and growing the cultures at different temperatures. Generally, protein expression needs to be induced at a specific OD (e.g. OD 0.6), which, with the traditional

method for monitoring cell culture growth by OD, requires the removal of a culture sample at

regular intervals and measurement of the sample in a bench-top spectrophotometer. This is a time consuming task, particularly if multiple cultures have to be monitored in parallel, and if an

incorrect OD is reached the experiment needs to be re-started.

With the use of the SMOD the user introduces the device into the culture when it is set up, this

eliminates the interruption of the culturing process to take manual measurements. Instead, the user obtains real-time information about the OD and temperature of the culture to their PC. This

becomes more and more convenient as the number of cultures being monitored increases.

Furthermore, because the SMOD takes measurements at a user-defined interval (as frequent as

every 10 minutes), protein expression can be induced at a very precise OD (Figure 1). This is particularly important if the recombinant protein is unstable, precipitates or has cleavage

problems.

An additional feature of the SMOD is that it also monitors temperature within the culture in real-time. This is important when the recombinant protein is particularly unstable because the

temperature at which the protein is expressed needs to be reduced (e.g. from 37°C to 20°C). In

this case the temperature in the culture needs to remain stable during the protein expression

period. The SMOD provides information about the change and stability of temperature in a culture during an expression experiment (Figure 1).

Copyright © 2016, Lifeonics Ltd.

Once the conditions for recombinant protein expression have been optimized the process is normally scaled up. The SMOD enables highly reproducible induction of expression at the

optimum determined OD and monitoring temperature and OD after induction, helping to ensure

consistency and aiding in diagnosis of failed cultures.

Conclusion

For anyone performing recombinant protein expression, finding optimum conditions and

maintaining the conditions constant once optimized is important. With the SMOD, automated OD

and temperature measurements are taken in the sample, enabling real-time continuous monitoring of vital culture parameters without interruption of the culturing process. This simplifies

the optimization of protein expression conditions and monitoring of these precise conditions once

they have been determined enabling the maximum yield of protein to be obtained.

Copyright © 2016, Lifeonics Ltd.

References: Kelly, K.D., Olive, L.Q., Hadziselimovic, A., Sanders, C.R. (2010). Look and See if it is Time to Induce Protein Expression in Eschericia coli Cultures. Biochemistry. 2010 Jul 6; 49(26): 5405–5407.

Figure 1. Using the SMOD for protein expression in Escherichia coli. To demonstrate the use of the SMOD to monitor E. coli cultures during induction and expression of proteins. E. coli BLIM cells were inoculated into LB medium at an OD600 of 0.05 and the growth was monitored with the SMOD at a 15 minute time interval until an optical density of 0.5 was reached (Black arrows) . The culture was grown at constant temperature (370C) with agitation (200rpm). Then the cultures were removed from the incubator to cool to room temperature before induction. Once the temperature had dropped, protein expression was induced by adding Arabinose (0.2% v/w, Red arrows), and the cultures were returned to a 200C incubator and incubated for 10 hours at constant temperature (200C) with agitation (200rpm). The Optical Density (OD600) and temperature measured by the two SMODs during the experiment is graphed.

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