Part:BBa_K4335004

mCherry

Contribution by 2023 iGEM Team SCIE-PearlDelta

Group: SCIE-PearlDelta iGEM 2023
Summary:
In this project, we used mCherry (BBa_K4335004) to connect with beta-2-Transferrin (bTF) which allows us directly to see whether the bTF protein is expressed via the color of the solution. Meanwhile, the mCherry also gives a fluorescence marker to the bTF protein, enabling the tracing in the membrane which could be measured by ELISA. In our experiments, we amplified the mCherry gene (BBa_K4335004) through PCR and linked it with using bTF (BBa_K4847000) GS-linker, and then connected this fusion gene fragments to the vector pET28a to to construct recombinant plasmids, pET28a-mCherry (BBa_K4847004) and pET28a-bTF-mCherry (BBa_K4847005) for subsequent experiments.

Documentation:

a. Usage and Biology:

The discovery of fluorescent proteins has played an important role in revealing the details of cell function[1]. mCherry is a red fluorescent protein, which is one of the reports for developing a universal gene expression method that uses 200 random nucleotides as regulatory sequences to drive the expression of coding sequences[2]. The efficiency of this method depends on genes and environment, but it typically produces 30% to 40% of successful protein expression in Escherichia coli[2] . However, when mCherry was used as a reporter gene, we observed fluorescence in approximately 65% of E. coli clones[3]

b. Characterization

We introduced mCherry to link our protein in order for the better visualization. After transforming the plasmids, pET28a-mCherry (BBa_K4847004) and pET28a-bTF-mCherry (BBa_K4847005) into E. coli BL21, IPTG inducing expression and protein purification were done for SDS-PAGE. In Figure 1 below, mCherry protein is about 30kDa and fusion protein bTF-mCherry is about 107kDa.

Figure 1. Electrophoresis gel of mCherry PCR and SDS-PAGE

On the test strip in Figure 2, we can see a red line with bared eyes. Under the spectrometer, we can observe the color of mCherry present in the red band, which is linked to bTF protein.

Figure 2. bTF-mCherry protein is observed on test strip and under spectrometer

References:

1. Chudakov D. M., Matz M. V., Lukyanov S., Lukyanov K. A. (2010). Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 90, 1103–1163.
2. Lale R., Tietze L., Nesje, J., Onsager I., Engelhardt K., Rückert C., et al. A universal method for gene expression engineering.
3. Fages-Lartaud, M., Tietze, L., Elie, F., Lale, R., & Hohmann-Marriott, M. F. (2022). mCherry contains a fluorescent protein isoform that interferes with its reporter function. Frontiers in bioengineering and biotechnology, 10, 892138.

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]