Difference between revisions of "Part:BBa K2916038"

Revision as of 20:02, 21 October 2019

Expression of EF-Tu in E.coli

This part is used for expression of Elongation Factor-Tu needed for the OnePot PURE cell-free system.

Usage and Biology

Used in OnePot PURE

Characterization

Expression and purification of EF-Tu

EF-Ti is one of the proteins we used for the OnePot PURE cell-free system. We expressed it in M15 E.coli strain using a pQE60 vector. The expression system has a T5 lac operator, RBS and a lambda t0 Terminator, enabling us to regulate the expression with IPTG.

Methods

EF-Tu was purified using our protocol . To test if the protein was actually expressed, we performed a SDS-PAGE that is presented below. On the left side we can see the results included in the initial OnePot PURE paper (Lavickova et al, 2019) while on the right (batch1_a,b and batch2_a,b) are the solutions we produced ourselves. (The procedure we followed and the conditions of the experiment can be found here).

Conclusion
EF-Tu has a molecular weight of around 43kDa, since there is a band shown at this level of molecular weight, we may assume that it is expressed. To verify the functionality of this protein we need to proceed with more experiments that would be mainly focused on the efficiency of the system.

OnePot PURE functionality test

To make sure that we have all the proteins in our OnePot PURE protein solution, and that they all function properly we need check if proteins can be expressed in our OnePot PURE cell-free system.

Methods

We expressed superfolding GFP following the protocol we designed in 10μl reactions, and measured the fluorescence on a plate reader at excitation wavelength of 535nm. We tested the expression using different concentrations of the sf GFP DNA template and also compared it with the fluorescence produced in PURExpress from NEB.

Conclusion
The expression was successful so we can confirm that EF-Tu exists in our protein solution and is also functioning properly.

Sequence and Features