Part:BBa_K2916014
Expression of AlaRS in E.coli
This part is used for expression of Alanyl-tRNA synthetase (AlaRS) needed in OnePot PURE cell-free system.
This part is used for expression of Alanyl-tRNA synthetase (AlaRS) needed in 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.
Alanyl-tRNA synthetase (AlaRS) attaches to Alanine (codons: GCU, GCC, GCA, and GCG)
In our part, besides the sequence encoding for the protein we also have a hexahistidine-tag to allow us purify the protein.
Usage and Biology
Used in OnePot PURE
Characterization
Expression and purification of AlaRS
AlaRS 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
AlaRS 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
AlaRS has a molecular weight of around 96kDa, 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.
Conclusion
The expression was successful so we can confirm that AlaRS 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 573
Illegal BglII site found at 780
Illegal BamHI site found at 746 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1184
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2584
Illegal BsaI site found at 2761
Illegal BsaI.rc site found at 1133
Illegal SapI site found at 1868
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