Replication methods and tools in high-throughput cultivation processes - recognizing potential variations of growth and product formation by on-line monitoring.
BACKGROUND
High-throughput cultivations in microtiter plates are the method of choice to express proteins from recombinant clone libraries. Such processes typically include several steps, whereby some of them are linked by replication steps: transformation, plating, colony picking, preculture, main culture and induction. In this study, the effects of conventional replication methods and replication tools (8-channel pipette, 96-pin replicators: steel replicator with fixed or spring-loaded pins, plastic replicator with fixed pins) on growth kinetics of Escherichia coli SCS1 pQE-30 pSE111 were observed. Growth was monitored with the BioLector, an on-line monitoring technique for microtiter plates. Furthermore, the influence of these effects on product formation of Escherichia coli pRhotHi-2-EcFbFP was investigated. Finally, a high-throughput cultivation process was simulated with Corynebacterium glutamicum pEKEx2-phoD-GFP, beginning at the colony picking step.


RESULTS
Applying different replication tools and methods for one single strain resulted in high time differences of growth of the slowest and fastest growing culture. The shortest time difference (0.3 h) was evaluated for the 96 cultures that were transferred with an 8-channel pipette from a thawed and mixed cryoculture and the longest time difference (6.9 h) for cultures that were transferred with a steel replicator with fixed pins from a frozen cryoculture. The on-line monitoring of a simulated high-throughput cultivation process revealed strong variances in growth kinetics and a twofold difference in product formation. Another experiment showed that varying growth kinetics, caused by varying initial biomass concentrations (OD(600) of 0.0125 to 0.2) led to strongly varying product formation upon induction at a defined point of time.


CONCLUSIONS
To improve the reproducibility of high-throughput cultivation processes and the comparability between different applied cultures, it is strongly recommended to use automated or manual liquid handling stations or, alternatively, multi-channel pipettes. Because of their higher transfer volume and hence precision in comparison to pin replicators, they reduce the variance of initial biomass concentrations. With respect to the results obtained, other methods to increase the comparability between parallel cultivations by compensating differences in biomass concentrations are required, such as using autoinduction media, fed-batch operation of precultures or on-line monitoring in microtiter plates combined with automated liquid handling.
AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
Robert
R
Huber
Thomas G
TG
Palmen
Nadine
N
Ryk
Anne-Kathrin
AK
Hillmer
Karina
K
Luft
Frank
F
Kensy
Jochen
J
Büchs
D001431:Bacteriological Techniques / Q000379:methods; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D048230:Corynebacterium glutamicum / Q000254:growth & development / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000254:growth & development* / Q000378:metabolism*; D007218:Industrial Microbiology / Q000295:instrumentation / Q000379:methods*
D003470:Culture Media