Part:BBa_K4335004

mCherry

mCherry is a red fluorescent protein. Fluorescent reporter genes for biological applications. We optimized its codon to express in Chlamydomonas reinhardtii more efficiently.[1]

BBa_K4335004 is the DNA of mCherry gene derived from mushroom coral and it is codon optimized for Chlamydomonas reinhardtii .

The advantage of mcherry over other fluorescent proteins is that its color and green fluorescent protein (GFP) are co-labeled, and mcherry also has excellent light stability over other monomeric fluorescent proteins.

Protein structure of mCherry.[Image origin]

It's believed that codon-choice have been conserved during evolution course and because not all tRNA are expressed equally, specially across species, a particular DNA sequence can be codon optimised to match the most prevalent tRNAs of the host cell, improving the efficiency of protein translation [1].

When we looked through the blast tool on the registry page, we found a part [BBa_K2136016] that was very similar to ours. This is also a codon optimized mCherry for Chlamydomonas reinhardtii.We compared the two sequences using snapgene and found that they were identical to our mcherry coding sequence.

Our optimized mCherry for Chlamydomonas reinhardtii.In addition to the optimization of codes, we also added a intron length of 145 bp .

The existence of introns in eukaryotes is an important characteristic that distinguishes them from prokaryotes. In higher organisms, introns have been reported to regulate gene expression at multiple levels. The main function of introns is to produce different exonic combinations through alternative splicing and then translate different proteins, thus increasing the complexity of the proteome. [2]

As a eukaryote, Chlamydomonas reinhardtii can express mCherry gene by adding intron.

The Top Blue Band is BBa_K2136016, the Middle Red Band is mCherry's coding sequence, and the bottom band is mCherry with introns.

Usage

We introduced the mCherry gene into plasmid [pTX2038] as its reporter gene. Using pCrRBCS2 as promoter and tCrRBCS2 as terminator, mCherry gene expression was realized.

Result

Plasmid construction

To validate the mCherry sequence in our vector, we designed mCherry-f and mCherry-R primers to verify our successful assembly.

mCherry and the position of primer targeting

Electrophoregram of amplification products,M is DNA Marker.

Functional Identification

We introduced the mCherry-containing plasmid pTX2038 into Chlamydomonas reinhardtii by electroporation and observed it using Fluorescence microscope.

Fluorescence excitation of positive clones transferred into pTX2038 vector compared with wild type mCherry, together with DIC field and chlorophyll excitation as fluorescence control.

Reference

[1] Codon usage and tRNA content in unicellular and multicellular organisms. T Ikemura. Mol Biol Evol (1985) 2 (1): 13-34.

[2]Parenteau, J. et al. Introns are mediators of cell response to starvation. Nature 565, 612–617 (2019).

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]