Difference between revisions of "Part:BBa K2213005"

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PduD(1-20)_mCherry_cgPPK2_His6

Figure 1: The overall architecture of the construct

The N-terminal PduD(1-20) domain (Figure 1) is sufficient to localise the part into a recombinant ethanolamine utilisation (Eut) or 1, 2 propanediol (Pdu) bacterial microcompartment (Jakobson et al., 2015). The mCherry allows monitoring the subcellular location of the part. The cgPPK2 domain encodes a soluble class II polyphosphate kinase from Corynebacterium glutamicim with a specific activity 30-fold greater than that of E. coli(Lindner et al., 2007;Kornberg and Simms, 1956). The part also contains a C-terminal hexa-histidine tag to allow purification with immobilised metal ion affinity chromatography.

The part was overexpressed in a BL21 (DE3) strain of E. coli under the control of a T7 promoter and purified to a certain degree (Figure 2)

(Figure 2:) SDS-PAGE analysis of the purification stages of the construct. Black arrows indicate bands with a molecular mass corresponding to the construct (66 kDa) and a heavier 70 kDa band that may correspond to Gro EL, which co-purified with the construct. Ladder: Precision Plus Protein™ (Bio Rad)

Expression optimisation using 'Design of Experiments'
Expression of this part downstream of a T7 promoter in the pNIC28-bsa4 expression vector in a BL21 (DE3) strain of E. coli was optimised in Luria Bertani broth using a 'Design of Experiments' based method, where the following input factors (and their estimated parameter limits in parentheses) were screened for their effect on protein yield:

OD600 at time of induction (0.2 - 0.8)

Final concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) at induction (0.1-1 mM)

Post induction growth period (4 - 24 hours)

Post-induction growth temperature (20 - 37°C)

JMP software from SAS was used to create a custom experimental design that required 20 initial experimental runs that would screen each of the factors and their two and three-way interactions for their effect on yield of PduD(1-20)_mCherry_cgPPK. The initial design assumed a linear relationship between the variables, so only tested the extreme values of the paramater limits.

The liquid cultures were created from a clonal culture or BL21 (DE3) cells containing a sequence verified pNIC28-bsa4:PduD(1-20)_mCherry_cgPPK vector and grown at 37 ˚C and 180 rpm until the desired OD600 was reached, at which point IPTG was added to the final concentration specified by the JMP design, and flasks were transferred to incubators at the specified temperatures for the specified growth periods. After fluorescence data from the first 20 runs was collected, a linear model was fit to the data using least-squares analysis with emphasis on effect screening (R²=0.98) which allowed us to identify factors that had a significant effect on yield (P<0.05)(Figure 3).

Figure 3: Effect summary of the screened factors and their two-way and three-way interactions on yield of the construct. Factors with a P value >0.05 were not considered to have a significant effect on yield.

The Effect screening from the first round suggested that all of the initial input factors had a significant effect on yield and all of them interacted with each other in a way that affected yield. The model was used to create an interaction profile (Figure 4) that would inform the design of the next round of optimisation.

References

Jakobson, C., Kim, E., Slininger, M., Chien, A. and Tullman-Ercek, D. (2015). Localization of Proteins to the 1,2-Propanediol Utilization Microcompartment by Non-native Signal Sequences Is Mediated by a Common Hydrophobic Motif. Journal of Biological Chemistry, 290(40), pp.24519-24533.
Lindner, S., Vidaurre, D., Willbold, S., Schoberth, S. and Wendisch, V. (2007). NCgl2620 Encodes a Class II Polyphosphate Kinase in Corynebacterium glutamicum. Applied and Environmental Microbiology, 73(15), pp.5026-5033.

Kornberg, A., Kornberg, S. and Simms, E. (1956). Metaphosphate synthesis by an enzyme from Escherichia coli. Biochimica et Biophysica Acta, 20, pp.215-227.
Sequence and Features