Difference between revisions of "Team:Tongji China/Record"

Plasmid Construction Parts (Week 1-6: June 5-July 10)

Week1

June 5

pUAST-3xHA transform into ''E.coli''.

Fruit fly screening and testing

We screened out the Drosophila in which our target gene was expressing stably.
To test whether our construction worked or not, we did some Molecular Biology (real-time PCR) and behavioral (male-male courtship and sexual preference change) experiments. Finally, we selected out the fruit fly which performed as expected and recorded their courtship behavior on video. Moreover, we designed experiments to compare the transgenic and wild-type fruit flies in terms of viability and fertility to test whether the reproductive function of transgenic Drosophila was normal.
Through eight experiments, we got the following results: [1] Dopamine levels are regulated by the temperature in the system and exhibit significant behavioral characteristics and [2] the reconstructed Drosophila is similar to the normal fruit fly in terms of survival and reproduction.

Model

We set up a mathematical model to predict whether our transformation of Drosophila can play a positive role in pest control.

Dipteran fly damage

Dipteran fly damage (stress on the animal) can be divided into direct and indirect hazards.

Direct hazards:
1) Bite, harassment: sucking mosquitoes, sand flies, gnats, midges and flies and other insects can be stabbed vampire, was bitten at itching, severe papules-like urticaria, affecting the normal life and production of animals.
2) Cause allergic reactions: arthropod salivary glands, secretions, excretions and shedding of the epidermis have all heterologous protein which can cause allergic reactions.
3) Parasitic: parasite larvae parasite can cause myiasis, for example, after bovine fly larvae parasitic in the bovine skin, the fur is perforated because of the larvae 'growth, which will lower its use value, at the same time, myiasis can also cause the decline in the quality of beef and the sharply drop in the amount of cattle milk.

Indirect hazards:
Many species of Diptera are used of vector insects in the transmission of pathogens between animals, pathogens, including bacteria, parasites, viruses and rickettsia and so on. Such as mosquito-borne malaria, filariasis, yellow fever and dengue fever, etc.
Chrysopa lucidum, Chrysanthemum, Chlamydomonas, Carassius auratus, Chlamydia trachomatis, Mosquitoes, gadfly and midges and other insect’ bites can make dairy cows infected with mastitis, which is one of the common breast disease during summer and dry milk.

Whether it is direct or indirect harm, the end result always affects the normal life and production of animals.

Fruit fly

The fruit fly Drosophila melanogaster is one of the most powerful genetically tractable model organisms. Work using Drosophila has made many valuable contributions to our understanding of animal development, behavior, and physiology and of human disease. While Drosophila is also a member of Dipteran fly.

UAS-Gal4

In yeast, GAL4 regulates the galactose metabolism, transcriptional activation of galactose utilization genes occurs when GAL4 binds to the upstream activation sequence (UAS) containing a varying number of a 17-mer repeat. GAL4 binds to DNA as a dimer through a Zn (2)-Cys (6) zinc finger. The N-terminal region mediates both dimerization and DNA binding and contains a nuclear localization signal, while an acidic C-terminal domain controls transcriptional activation. GAL4 directly interacts with the Tra1 component of the SAGA complex, recruiting Mediator and the general transcriptional machinery to initiate transcription. This ability to activate transcription is retained when GAL4 is expressed in other species including plants, human cell lines, zebrafish, and Drosophila.

Gal80 and Gal80ts

The activity of GAL4 can be repressed by physical interaction with the yeast GAL80 protein. A dimer of GAL80 binds to the C-terminal ends of the GAL4 dimer so that, while it can still bind to a UAS sequence, it can no longer activate transcription. This interaction of GAL4 and GAL80 can be taken advantage of to refine the expression pattern of GAL4-dependent transgenes.
Temporal specificity can be achieved using a temperature sensitive allele of GAL80. The Temporal and Regional Gene Expression Targeting (TARGET) system takes advantage of a variant of GAL80 that while ubiquitously expressed under control of the tubulin 1α promoter is only active at permissive temperatures.
Thought to be the result of a single glycine to arginine substitution at amino acid 203 of GAL80, GAL80ts is unable to bind GAL4 at restrictive temperatures above 29 °C but retains its repressive function at the permissive temperature of 18 °C. Controlling the activity of GAL80ts through temperature shifts provides temporal control of GAL4-dependent transgene expression. Limiting expression to defined temporal windows can help to define critical periods for the effects of misexpression or rescue experiments. Temperature- sensitive alleles of GAL4 itself have been generated and tested in Drosophila, however the ease of combining tubGAL80ts with already established GAL4 drivers cause the TARGET system to be more commonly used.

Christian Dahmann. Drosophila Methods and Protocols 2nd Edition. New York: Humana Press, 2016: 33-38.