We analyzed existing parts BBa_K912000 and BBa_912001 using OD. Please see the results in parts regigsry page in Parts page.
Until the Wiki freeze, we tried to construct plasmid with the insert we designed and managed to combine plasmid with INSERT 1, 2 or 3(we couldn't insert INSERT 4, which is a control of the INSERT 2 or 3).
To assay our construct, we cultured E. coli with IPTG induction, purified the crude extract by the His-tag and condensed them.
When the expressed protein (both INSERT 2 and 3) were cut at the signal peptides, the molecular weight becomes 52367.76 or 48575.69 respectively.
The figure bellow is the result of SDS-PAGE. We applied purified INSERT 2 (left) and INSERT 3 (right, not visible)and found that the band of INSERT 2 is at the correct place.
We assumed two possibility that explain why we couldn't get the band of INSERT 3; the concentration of protein is too small, the His-tag didn't interacted with the Nickel column and flowed through.
To determine the reason why we couldn't see the band, we conducted phytase activity assay with the crude extract, purified and condensed products, and the flow through of purification process.
As the table and figure below shows, we can see high phytase activity in the flow through and concluded that His-tag didn't interacted with the Nickel, probably because the His-tag was not exposed outside. Simultaneously, we can assume that self-Assembling Peptides (SAPs) helped His-tag being exposed and interacting with Nickel.
We applied crude extract of INSERT 2 (left) and INSERT 3. (right). The difference of mobility between INSERT 2 and 3 is very small but may be slightly different. (Difficult to judge)
We assayed thermostability of several kind of phytase; Citrobacter braakii and E. coli phytase from Northwestern 2012 and Klebsiella pneumoniae (our original)
Though the degree of deactivation is different according to kind of phytase, the characteristic that remaining activity after 1 hour heat shock drops is common.
From the comparison between INSERT 2 and 3, we can see that activity of phytase from insert 3 drops more drastically than that of from INSERT 2, suggesting that SAPs will improve thermostability of phytase.
- We confirmed the deactivation of phytase due to heat shock.
- We found the improvement of thermostability by SAPs.
- Exposure of the His-tag due to SAPs is suggested.
- We couldn't construct the control part (expression of phytase only) and couldn't assay the function of cysteine module.