Team:SUSTech Shenzhen/Part Collection

Team SUSTC-Shenzhen

Parts_Collection

Contribution


Category Name Description
Reporter <partinfo>BBa_K2492000</partinfo> GFP optimized with three introns for C.elegans
Coding <partinfo>BBa_K2492001</partinfo> CoCHR
Reporter <partinfo>BBa_K2492002</partinfo> Mcherry optimized with three introns for C.elegans
Coding <partinfo>BBa_K2492003</partinfo> GEM-geco Caucium indicator
Promoter <partinfo>BBa_K2492004</partinfo> ord-10

Part: BBa_K2492000

Modified GFP with three introns which can help well expression of GFP in C.elegans. In our group, we use it to indicate the location of neuron AWB in C.elegans. We integrated str1::CoCHR::GEM-GECO::GFP transgene in C.elegans by microinjection. Modified GFP will be highly expressed in AWB driven by promoter str1 (no biobrick part).

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Part: BBa_K2492001

The central part of our project. Part BBa_K2492001 contains CoCHR(Chloromonas oogama channelrhodopsin) [1].

In our project, we hope to investigate how olfactory messages are transmitted by the neuron systems. In order to decrease delay or response comparing to chemical stimulus, we choose optogenetic techniques. Since C.elegans do not have sense to light, we express channelrodopsins in pairs of neuron as light receptors. CoCHR is the channelrodopsin in transgenic AWA. In order to express CoCHR in AWA specifically, we express CoCHR through the linkage to the AWA cell-specific promoter odr-10. Thus, when we shed ~470nm excitation light to transgenic C.elegans, the activated AWA is able to cause obvious response such as body swimming towards. While wild-type C.elegans not behave abnormally under the same stimulation. Whether the certain neuron is activated or not is indicated by GEM-GECO, a calcium indicator. In short, we established a light-sensed AWA controlling system, so as to utilize this part to manipulate the activation of AWA, establish the response to input stimulation. The merit of CoCHR is its sensitivity to light which is five-times than formal ChR2. Thus, the low intensity requirement prevents the cross-activation of Crimson(Chlamydomonas noctigama channelrhodopsin). Chrimson is another integrated channelrhodopsin in AWB. (Moreover, in order to indicate where the target neurons are. We also construct a fusion reporter protein called mCherry. At the same time, the fusion protein is allowed to estimate the expression level of CoCHR.)

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Part: BBa_K2492002

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[2]. We can obtain ord-10::Chrimson::GEM-GECO::mCherry transgenic offsprings microinjection. MCherry driven by promoter ord-10(Part:BBa_K2492004) 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.

Part: BBa_K2492003

The central part of our project. Part BBa_K2492003 contains GEM-GECO(blue-green emission ratiometric GECO). GEM-GECO fluoresces green without Ca2+ but blue with Ca2+ [3]. GEM-GECO is used as a calcium ion indicator, so as to visualise neuron activity(AWA for instance). In recent advances, calcium is a universal indicator for neuron excitation, because calcium ions are able to influx at action potential. GEM-GECO is capable of monitoring and responding to the concentration change of calcium ions due to the existence of fluorescence resonance energy transfer between two chromophores inside of the functional domain. The reason why GEM-GECO enables the response of disparate calcium ion concentration is that in the Ca2+-free state, the chromophore of this blue-green emission ratiometric GECO (GEM- GECO) undergoes excited-state proton transfer (ESPT) and emits from the lower-energy (green fluorescent) anionic form. [4], while in the calcium ions bound state,two chromophores are less compact and photon transfer is blocked, resulting in blue fluorescent.

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Part:BBa_K2492004

Part BBa_K2492004 contains odr-10. It is a section of genomic DNA acts as a promoter. In C.elegans, ODR-10 encodes a large family of seven transmembrane olfactory receptor genes[5].which is specifically expressed in neuron AWA and affect chemotaxis to the volatile diacety. POrd-10 is a section of genemoic DNA in front of Ord-10 genes promoting expression of Ord-10 genes[6].

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References

  1. ↑ Schild, L. C., & Glauser, D. A. (2015). Dual color neural activation and behavior control with chrimson and cochr in caenorhabditis elegans. Genetics, 200(4), 1029-34.
  2. ↑ S.Redemann. (2011). codon adaptation– based control of protein expression in C. elegans, Nature Methods, Vol.8 No.3 250-254.
  3. ↑ Yanli Wang et al. (2014). Excited State Structural Events of a Dual-Emission Fluorescent Protein Biosensor for Ca2+ Imaging Studied by Femtosecond Stimulated Raman Spectroscopy, The Journal of Physical Chemistry B.
  4. ↑ Yongxin Zhao et al. (2011). An Expanded Palette of Genetically Encoded Ca2+ Indicators, Science, Vol. 333, 1888-1891.
  5. ↑ Sengupta P., Chou J.H., Bargmann C.I.Cell 84:899-909(1996)
  6. ↑ Emily R. et al. (1997). Reprogramming Chemotaxis Responses: Sensory Neurons Define Olfactory Preferences in C. elegans, Cell, Vol. 91, 161–169.


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