COE Study Abroad Project Report

 

Report on the 6-week stay in Macnabfs laboratory at Yale University

Nao Moriya, Master's course student



1. Report on the 6-week stay in Macnabfs laboratory at Yale University

2. Nao Moriya

3. Protonic Nanomachine Group

4. Department of Molecular Biophysics & Biochemistry, Yale University

5.
Purpose of the trip
To learn how to produce mutants which contain site-directed mutations during the 6-week stay in Macnabfs laboratory at Yale University, which has excellent techniques of gene manipulation.

Details
Bacterial flagellum consists of three parts; the basal body, the hook and the filament. The hook with a defined length of 55 nm functions as a universal joint to transmit the torque generated by the basal body (motor) to the helical filament (screw) smoothly. The aim of my study is to investigate the correlation between the hook structure and its function and to understand the mechanism that controls the hook length to be about 55 nm.
I isolated about 20 mutants of Salmonella flgE (the gene of the hook protein) that showed some impairment in motility. All of them contained a couple of random mutation sites, and therefore, I needed to produce mutants that contain point mutation site corresponding one of those sites or combinations of sites in order to analyze their phenotypes and the roles of those sites. Fortunately I had an opportunity to learn how to produce mutants that contain point mutations during the 6-week stay in Macnabfs laboratory at Yale University, which has excellent techniques of gene manipulation.
During my 6-week stay, I also tried to construct some plasmids for other members in our group who want to analyze the DNA sequences of flgE pseudorevertants of the mutants I isolated.

A. Production of point mutants
By PCR with two complementary primers that contain mutations at target sites, one can obtain a plasmid with a gene with mutations at any desired sites. This technique is called site-directed mutagenesis. I tried to produce ten patterns of mutants, and I was able to isolate 9 of them.

B. Construction of plasmids for overexpression of flagellar proteins
I tried to construct four types of plasmids for overexpression of flagellar proteins. This work was to help other members of our group. Target genes were amplified by PCR by using Thermotoga chromosome DNA as a template and cloned into plasmids. I was able to construct two plasmids but failed to construct other two.
@@
C. DNA sequence analysis of pseudorevertants
I did DNA sequencing of 19 pseudorevertants of flgE mutants out of 40 that I isolatedEleven of them had intragenic 2nd-site mutations, but no mutation sites were found in the rest, for which I will find extragenic mutation sites.
@@
What I gained from this trip
I learned various experimental techniques such as site-directed mutagenesis, which will be very useful for my future experimental work. I also had many good advice from the members of Macnab laboratory.
This trip was my first experience to to experimental work in a laboratory in a foreign country. This experience certainly broadened my view of many things. I realized how important it is to have active discussion daily. I also leaned many important things from their sincere and serious attitudes to the research work.

Prospective and future plan
I will analyze the effects of mutations of the mutants I produced and then make mutants containing combinations of mutation sites to see these the effect of combination as well.
@ I will also analyze the DNA sequence of flgD (hook capping protein) of mutants that did not have intragenic 2nd-site mutations to see whether or not I can find the 2nd-site mutations in flgD.
To advance my study including these 2 experiments, I would like
@) to study the mutation sites on the atomic model of the hook built by F. Samaty in our group,
A) to construct the model of hook protein including the region that could not be seen in the crystal structure,
B) to understand the mechanism by which the hook length is controlled to be about 55nm.