Difference between revisions of "Part:BBa K2213005"

Latest revision as of 04:00, 2 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) and kinase activity was qualitatively confirmed using the Promega ADP-Glo™ Kinase assay kit ( Figure 3).

Figure 3 Polyphosphate Kinase Activity Determined By the ADP Glo TM Kinase Assay Kit (Promega) With 0.2 mg/ml of Eluted Protein

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 (LB) 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 in shake flasks 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 4).

Figure 4: 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 5) that would inform the design of the next round of optimisation.

Figure 5: The interaction profile of the input factors, maximised for yield. OD600 at induction consistently correlated with higher yields. The profile also suggests that optimal conditions for expression of PduD(1-20)_mCherry_cgPPK lie close to 20°C and a post-induction growth period of 24 hours or more.


A second round of optimisation explored different paremater limits for:

Final concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) at induction (1 - 10 mM)

Post induction growth period (24 - 48 hours)

Post-induction growth temperature (16 - 24°C)

OD600 at induction was not screened in this round because it gave a robust positive correlation with yield, so was fixed to 0.8. 20 experiments that explored the above factors within their parameters were designed and performed. A linear model was fit to the data using least squares analysis with emphasis on effect screening (R² = 0.80) and identified post-induction growth temperature and post-induction growth period as the two most significant factors affecting yield of the construct within the new parameters (Figure 6). The interaction profile for this data suggested that the optimal concentration of IPTG at induction lay close to 1 mM (Figure 7). These two factors were plotted against yield of PduD(1-20)_mCherry_cgPPK in a three-dimensional surface plot (Figure 8).

Figure 6: Effects summary for the second round of optimisation experiments. The most significant factors within the new parameters are post-induction incubation temperature and post-induction growth period (harvest time).


Figure 7: The interaction profile for factors screened in the second round of expression optimisation.

Figure 8: A surface plot mapping the effect of post-induction growth temperature and post-induction growth period (harvest time) against yield of PduD(1-20)_mCherry_cgPPK (relative fluorescent units).


Based on the results of 40 individual experiments, the predicted optimal growth conditions to yield high levels of PduD(1-20)_mCherry_cgPPK under a T7 promoter in a BL21 (DE3) cell strain in LB broth are:
Induce cells at OD600 = 0.8 with 1 mM of IPTG. Incubate for a further 48 hours at 20 - 24°C before harvest.

An attempt to express and purify the PduD(1-20)_mCherry_cgPPK construct under optimal conditions revealed that most of the enzyme localised to the insoluble cell debris fraction after repeated attempts to disrupt the cells (Figure 9) and very little soluble PduD(1-20)_mCherry_cgPPK could be purified without using denaturing conditions.

Figure 9: SDS-PAGE analysis of the purification steps of PduD(1-20)_mCherry_cgPPK expressed under the predicted optimal conditions revealed that the most of the construct was retained in the insoluble fraction.


Assessing the thermal stability of cgPPK2 and PduD(1-20)-cgPPK2-mCherry
The heat stability of these constructs was determined through the use of a Thermal Shift Assay of the constructs cgPPK2_His6 (https://parts.igem.org/Part:BBa_K2213003) and PduD(1-20)_mCherry_cgPPK2_His6 (https://parts.igem.org/Part:BBa_K2213005).

Figure 10: A thermal shift assay of the constructs BBa_K2213003 and BBa_K2213005 using the dye sypro orange. Readings were taken at 0.4ºC intervals, 30 seconds after the solution had maintained that temperature. The samples were tested in duplicate before being normalised, so that the highest reading of each run was equal to 1; the mean was then plotted. Error bars showing the standard deviation are also shown.

From figure 10, cgPPK2His6 showed maximum change in denatured protein between 29.8ºC - 30.6ºC; whereas the tag-mcherry-ppk construct showed peaks at 32.2ºC - 32.4ºC. This suggests that the addition of a PduD tag and mCherry protein slightly increases the heat stability of the cgPPK2 protein. The tag-mCherry-PPK construct also consistently showed a second peak at 95.2ºC. Using Imperial2011's experience with BBa_I13521 (https://parts.igem.org/Part:BBa_I13521:Experience) we believe this peak is likely to be caused by the unfolding of the mCherry domain. Further research also seems to support this hypothesis. (Probing the StaBIlity of Fluorescent Proteins by Terahertz Spectroscopy, 2014)

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