Part:BBa_K2916038
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1292
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 389
Illegal AgeI site found at 794 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 539
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