GFP-fusioner med TRPA1 från Anopheles gambiae för överuttryck i Pichia pastoris
(2023) KEMK10 20222Department of Chemistry
- Abstract
- Animals monitor environmental ques by translating them into electrical signals through a depolarization of the electric potential over the cell membrane of nerve cells. A way to initiate the depolarization is through activation of receptors that are ion channels. These channels can facilitate the diffusion ions across membranes driven by the differences in concentration and electrical charge on each side of the membrane. TRP is a family of ion channels present in cell membranes. In particular, TRPA1 is an ion channel on the plasma membrane of eukaryotes and it is activated by harmful chemical substances which are causing pain and itch. However, this peculiar ion channel in malaria mosquitoes (Anopheles gambiae; Ag) is crucial for its host... (More)
- Animals monitor environmental ques by translating them into electrical signals through a depolarization of the electric potential over the cell membrane of nerve cells. A way to initiate the depolarization is through activation of receptors that are ion channels. These channels can facilitate the diffusion ions across membranes driven by the differences in concentration and electrical charge on each side of the membrane. TRP is a family of ion channels present in cell membranes. In particular, TRPA1 is an ion channel on the plasma membrane of eukaryotes and it is activated by harmful chemical substances which are causing pain and itch. However, this peculiar ion channel in malaria mosquitoes (Anopheles gambiae; Ag) is crucial for its host seeking behavior, as it operates on environmental temperature gradients. Therefore, to simplify detection and thus facilitate further studies on AgTRPA1, clones encoding sequences of AgTRPA1 fused with a green fluorescent protein (GFP) will be constructed in this project. A PCR product encoding the GFP-tag with following His-tag at its C-terminal amplified from the plasmid pPICZA- eGFP will be cut with the restriction enzymes Esp3I and BamHI. Furthermore, the DNA fragment encoding the GFP-tag will later be ligated into plasmids encoding full length and truncated versions of AgTRPA1, cut open by NotI and BamHI, and then be transformed into competent Escherichia coli cells. The resulting plasmids will then be prepared from the isolated transformants, verified by restriction enzyme digestion and then sent to sequencing. Overall, the project proceeded according to expectations. As a result, three clones could successfully be verified by sequence data. The data confirmed a properly ligated fragment encoding the GFP-tag in frame with the encoding sequence of AgTRPA1 and an intact BamHI site. To continue this project, the next step will be to put these promising sequences inside the yeast Pichia pastoris, express, isolate and hopefully determine the structure of AgTRPA1. The final goal is to create a molecule that works as a repellent that activates only AgTRPA1 without affecting the human orthologous receptor. (Less)
- Popular Abstract (Swedish)
- TRPA1, är en smärtreceptor på cellmembran vilken aktiveras av skadliga kemiska ämnen, som t.ex. allylisotiocyanater från senap, pepparrot och wasabi och därav kallas TRPA1 även för wasabi-receptorn. Smärta och klåda är tydliga symptom på högre aktivitet hos receptorn. Malariamyggan (Anopheles gambiae), använder denna receptor för att känna av skillnader i miljötemperaturer och receptorn spelar därav stor roll för myggan när den söker byte. I denna studie har grönt fluorescerande protein (GFP) inkorporerats med malariamyggans TRPA1. Tre kloner av GFP-fusioner med malariamyggans TRPA1 har framgångsrikt kunnat verifieras med sekvensdata, två fullängdsplasmider och en trunkerad plasmid. Genom att följa fluorescensen kan de kloner som har högst... (More)
- TRPA1, är en smärtreceptor på cellmembran vilken aktiveras av skadliga kemiska ämnen, som t.ex. allylisotiocyanater från senap, pepparrot och wasabi och därav kallas TRPA1 även för wasabi-receptorn. Smärta och klåda är tydliga symptom på högre aktivitet hos receptorn. Malariamyggan (Anopheles gambiae), använder denna receptor för att känna av skillnader i miljötemperaturer och receptorn spelar därav stor roll för myggan när den söker byte. I denna studie har grönt fluorescerande protein (GFP) inkorporerats med malariamyggans TRPA1. Tre kloner av GFP-fusioner med malariamyggans TRPA1 har framgångsrikt kunnat verifieras med sekvensdata, två fullängdsplasmider och en trunkerad plasmid. Genom att följa fluorescensen kan de kloner som har högst halt av fusionsproteinet lättare identifieras och dess upprening optimeras. Nästa steg i projektet kommer vara att tillverka proteinet i jästen Pichia pastoris, som används för att producera proteiner i stora mängder. Slutgiltligt mål i framtiden är att få bättre förståelse för hur proteinet fungerar i myggan och om det är möjligt att bekämpa sjukdomar som myggan sprider genom att designa en molekyl som fungerar som en repellent som aktiverar myggans receptor utan att påverka den motsvarande smärtreceptor i människa. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9106448
- author
- Mazzeo, Lisa LU
- supervisor
- organization
- course
- KEMK10 20222
- year
- 2023
- type
- M2 - Bachelor Degree
- subject
- keywords
- Biochemistry, TRP, AgTRPA1, GFP
- language
- Swedish
- id
- 9106448
- date added to LUP
- 2023-01-12 08:01:52
- date last changed
- 2023-01-12 08:01:52
@misc{9106448,
abstract = {{Animals monitor environmental ques by translating them into electrical signals through a depolarization of the electric potential over the cell membrane of nerve cells. A way to initiate the depolarization is through activation of receptors that are ion channels. These channels can facilitate the diffusion ions across membranes driven by the differences in concentration and electrical charge on each side of the membrane. TRP is a family of ion channels present in cell membranes. In particular, TRPA1 is an ion channel on the plasma membrane of eukaryotes and it is activated by harmful chemical substances which are causing pain and itch. However, this peculiar ion channel in malaria mosquitoes (Anopheles gambiae; Ag) is crucial for its host seeking behavior, as it operates on environmental temperature gradients. Therefore, to simplify detection and thus facilitate further studies on AgTRPA1, clones encoding sequences of AgTRPA1 fused with a green fluorescent protein (GFP) will be constructed in this project. A PCR product encoding the GFP-tag with following His-tag at its C-terminal amplified from the plasmid pPICZA- eGFP will be cut with the restriction enzymes Esp3I and BamHI. Furthermore, the DNA fragment encoding the GFP-tag will later be ligated into plasmids encoding full length and truncated versions of AgTRPA1, cut open by NotI and BamHI, and then be transformed into competent Escherichia coli cells. The resulting plasmids will then be prepared from the isolated transformants, verified by restriction enzyme digestion and then sent to sequencing. Overall, the project proceeded according to expectations. As a result, three clones could successfully be verified by sequence data. The data confirmed a properly ligated fragment encoding the GFP-tag in frame with the encoding sequence of AgTRPA1 and an intact BamHI site. To continue this project, the next step will be to put these promising sequences inside the yeast Pichia pastoris, express, isolate and hopefully determine the structure of AgTRPA1. The final goal is to create a molecule that works as a repellent that activates only AgTRPA1 without affecting the human orthologous receptor.}},
author = {{Mazzeo, Lisa}},
language = {{swe}},
note = {{Student Paper}},
title = {{GFP-fusioner med TRPA1 från Anopheles gambiae för överuttryck i Pichia pastoris}},
year = {{2023}},
}