Part:BBa_K2916027

Expression of PheRS in E.coli

This part is used for expression of Phenylalanyl-tRNA synthetase needed for the PURE and OnePot PURE cell-free systems.

Usage and Biology

Used in OnePot PURE

Expression and purification of PheRS

PheRS is one of the proteins we used for the OnePot PURE cell-free system and is separated in PheRS-α and PheRS-β. We expressed both of them in the same M15 E.coli strain using a pQE30 vector. The expression system has a T5 lac operator, RBS and a lambda t0 Terminator, enabling us to regulate the expression with IPTG.

Methods

PheRS 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).

Figure 1: SDS-PAGE of OnePot PURE protein solution.

Conclusion
PheRS-α has a molecular weight of around 36kDa while PheRS-β has a molecular weight of around 87kDa, but even though we cannot be absolutely sure if the bands shown are only due to them, we may assume that both are expressed. To verify the existence and 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.

Figure 2: sf GFP expression using 10nM DNA template.

Figure 3: sf GFP expression using 5nM DNA template.

Figure 4: sf GFP expression using 2.5nM DNA template.

Figure 5: Comparison between OnePot PURE and PURExpress at saturation.

Conclusion
The expression was successful so we can confirm that both PheRS-α and PheRS-β exist in our protein solution and are also functioning properly.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2533
  Illegal BamHI site found at 140
  Illegal BamHI site found at 1881
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1271
  Illegal AgeI site found at 1264
  Illegal AgeI site found at 2186
  Illegal AgeI site found at 3038
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2984
  Illegal SapI.rc site found at 307
  Illegal SapI.rc site found at 2960