Difference between revisions of "Part:BBa K2213005"

Revision as of 23:54, 1 November 2017

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). Ladder: Precision Plus Protein™ (Bio Rad)

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 using a 'Design of Experiments' based method, where the following input factors were screened for their effect on protein yield: OD600 at time of induction Final concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) at induction Post induction growth period Post-induction growth temperature

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