Part:BBa_K2916018

Expression of CysRS in E.coli

This part is used for expression of Cysteinyl-tRNA synthetase (CysRS) needed for the OnePot PURE cell-free system.

Biology

Transfer RNA (tRNA) is the molecule that enables the Genetic Code contained in the nucleotide sequence of a messenger RNA (mRNA) molecule to be translated into the amino acid sequence of a polypeptide chain. This function is catalysed by a group of enzymes called aminoacyl-tRNA synthetase (aaRS) which attach the appropriate amino acid onto its tRNA. This group consists of 20 different types of aminoacyl-tRNA synthetases, one for each amino acid of the genetic code. Those enzymes, in presence of Adenosine triphosphate (ATP) and Amino Acids, produces aminoacyl-tRNA (tRNA charged with an Amino Acid) that can be used by the ribosome to transfer the amino acid from the tRNA to the polypeptide being synthesised, according of course to the genetic code. Aminoacyl-tRNA therefore play a major role in RNA translation.

Cysteinyl-tRNA synthetase (CysRS) attaches to Cystein (codons: UGU and UGC)

In our part, besides the sequence encoding for the protein we also have a hexahistidine-tag to allow us purify the protein.

Usage

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

Expression and purification of CysRS

CysRS is one of the proteins we used for the OnePot PURE cell-free system. We expressed it in BL21(DE3) E.coli strain using a pET21a vector. The expression system has a T7 promoter, a lac operator, RBS and a T7 Terminator, enabling us to regulate the expression with IPTG.

Methods

CysRS 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
CysRS has a molecular weight of around 52kDa, 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 CysRS exists in our protein solution and is also functioning properly.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1533
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1087
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 953