Part:BBa_K2916042

Expression of RF3 in E.coli

This part is used for expression of Release Factor 3 (RF3) needed for the OnePot PURE cell-free system.

Usage and Biology=

Release factors are proteins that function during the third and final step of the translation, the termination. When they recognize the termination or stop codon in an mRNA sequence, they release new peptides from the ribosome. In the standard genetic code, there are three mRNA stop codons: UAG ("amber"), UAA ("ochre"), and UGA ("opal" or "umber")

In our project we used RF3 as a part of the protein solution needed for OnePot PURE cell-free system produced with the method of gravity flow affinity chromatography, as described in the protocol we designed.

Expression and purification of RF3

RF3 is one of the proteins we used for the OnePot PURE cell-free system. We expressed it in 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

RF3 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
RF3 has a molecular weight of around 60kDa, but even though we cannot be absolutely sure if the band shown is only due to it, we may assume that it is 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 RF3 exists in our protein solution and is also functioning properly.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 979
  Illegal AgeI site found at 1040
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1203