Part:BBa_K2005050:Experience

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Group: iGEM17_SUSTech_Shenzhen

Author: iGEM17_SUSTech_Shenzhen

Summary:We found that the part BBa_K2005050 is mCherry submitted by iGEM16_Vanderbilt. However, this mCherry is not designed for C.elegans. Thus, we got the sequence and improved it with C.elegans codon adaptor[1]. Compared with original mCherry, new synthesis mCherry sequence is modified in: a.adding three introns b.some codons modified The mCherry part we used now is optimized for C.elegans and perform excellently observed under confocal microscope. We added the figure to part page The whole neuron with dendrite can be observed distinctly. The branch of dendrite near the mouse can be observed as well(Figure). Universal mCherry coding sequence of three introns for capacity of expression in C.elegans. MCherry is a commonly used reporter. Since ordinary mCherry may not have high expression in C.elegans, artificial introns are inserted in the constructed plasmid[1]. We can obtain ord-10::CoCHR::GEM-GECO::mCherry transgenic offsprings microinjection. MCherry driven by promoter ord-10(Part:BBa_K2492004[1]) indicates the position of neuron AWA. Moreover the fluorescence of mCherry aids the imaging for the neuron AWA pattern. The intensity of fluorescence reveals the expression level approximately. [1]:S.Redemann. (2011). codon adaptation– based control of protein expression in C. elegans, Nature Methods, Vol.8 No.3 250-254.

mcherry expressed in AWA

Test for GFP: localize the neuron-AWA

We successfully ligated the Pord-10::CoCHR::GEM-GECO::mCherry and injected the plasmids into C.elegans through microinjection. This picture was one of our stable inheritance strains. We used 543nm to excite AWA and observe at 610nm wavelength through confocal. We successfully observed two pairs of cherry red slender neurons on the anterior region of C.elegans. Comparing to the normal localisation of AWA, we indicated that the fluoresecence of mcherry appeared in AWA. Thus we successfully used mcherry to localise the position of target neuron. And mcherry is characterized by our experiment.