Replication methods and tools in high-throughput cultivation processes - recognizing potential variations of growth and product formation by on-line monitoring

Abstract

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.

Figure 1

Scheme of a typical high-throughput cultivation process. After transformation, the clones are plated and grown on agar. Colonies are transferred with sterile toothpicks to a preculture-MTP and cultivated. Glycerol is added and the MTP is frozen (cryoculture). After thawing and mixing, liquid is transferred from the cryoculture-MTP to a new preculture-MTP. This MTP is incubated and subsequently a new main culture-MTP is inoculated with liquid from the preculture-MTP. At a defined point of time, inducer is added to the main culture to induce protein expression.

Figure 2

Influence of different replication tools on growth of 96 cultures of E. coli PR02. Replication tools: A) 8-channel pipette, thawed and mixed cryoculture. B) Disposable plastic replicator, thawed and mixed cryoculture. C) Steel replicator, fixed pins, thawed and mixed cryoculture. D) Steel replicator, fixed pins, thawed cryoculture, not mixed. E) Steel replicator, fixed pins, frozen cryoculture. F) Steel replicator, spring-loaded pins, frozen cryoculture. Experimental conditions: TB medium with 5 g/L glycerol, filling volume per well 200 μL, shaking frequency 950 rpm, shaking diameter 3 mm, temperature 37°C, measurement with BioLector.

Figure 3

Influence of different initial biomass concentrations on growth and product formation of E. coli BL21(DE3) pRhotHi-2-EcFbFP. A) Scattered light intensity (mean of eight parallel wells per condition). B) EcFbFP fluorescence intensity (mean of eight parallel wells per condition). Vertical dashed line: time of induction with 0.1 mM IPTG after 4.9 h. Experimental conditions: MDG medium, filling volume per well 200 μL, shaking frequency 950 rpm, shaking diameter 3 mm, temperature 37°C, measurement with BioLector.

Figure 4

Simulating a typical high-throughput cultivation process: effect on growth and product formation of Corynebacterium glutamicum pEKEx2-phoD-GFP. Transformed genetically identical C. glutamicum clones were grown on agar-plate. Sixty-two colonies from this plate were transferred to a MTP with toothpicks and cultivated over night. Glycerine was added to the MTP which was then frozen at -20°C. The cryoculture-MTP was thawed and mixed. Subsequently, 10 μL of each well were transferred to 190 μL medium in a preculture MTP. After cultivation over night 10 μL of each well were transferred to 190 μL medium in a main culture MTP. The following cultivation with induction was monitored with the BioLector and is shown above. A) Scattered light intensity. B) GFP fluorescence intensity. Vertical dashed line: time of induction with 0.5 mM IPTG after 5.25 h. Experimental conditions: modified Eikmanns mineral medium, filling volume per well 200 μL, shaking frequency 995 rpm, shaking diameter 3 mm, temperature 37°C, measurement with BioLector.