M.

Sc (MICROBIOLOGY) 2 YEAR PROGRAM; 1 SEM

MICROBIOLOGY LAB -I (19M25BT111)
Vikas sharma
20915002

EXPERIMENT- 2C
AIM- To identify unknown bacterial species using 16s rrna gene sequence.

Materials required:
Computer with internet, 16s rRna sequence of unknown bacteria.

Theory:
The use of 16 rrna gene sequence to study bacterial phylogeny and taxonomy
has been by further most common housekeeping genetic marker used for a
number of reasons include:
1. Its presence in almost all bacteria after existing as a multigene family or
operons.
2. The function of the 16s rRna gene over time has not changed suggesting
that random sequence changes are more accurate measure of time.
3. The 16s rRna gene is large enough for informatics purposes. It has been
demonstrated that 16s rRna gene sequence data on an individual strain
with a nearest neighbour exhibiting similarity score of <97% represents a
new species.
BLAST- BLAST (basic local alignment search tool) is an algorithm and program
for comparing primary biological sequence information, such as the amino-acid
sequences of proteins or the nucleotides of DNA and/or RNA sequences. A
BLAST search enables a researcher to compare a subject protein or nucleotide
sequence (called a query) with a library or database of sequences, and identify
database sequences that resemble the query sequence above a certain
threshold.
MSA viewer- Multiple sequence alignment is a tool used to study closely related
genes or proteins in order to find the evolutionary relationships between genes
and to identify shared patterns among functionally or structurally related genes.
FASTA- the FASTA format is a text-based format for representing either
nucleotide sequences or amino acid (protein) sequences, in which nucleotides or
amino acids are represented using single-letter codes. The format also allows for
sequence names and comments to precede the sequences.
Clustal Omega is a new multiple sequence alignment program that uses seeded
guide trees and HMM profile-profile techniques to generate alignments between
three or more sequences.

Procedure:
A. Use unknown 16s rRna sequence to identify
1. Select the word file and copy the unknown sequence from it.
2. Open the BLAST web site select blastn.
3. Paste the sequence in the Query Sequence and then search.
4. Select the showing results and open in the MSA viewer.
5. Download the FASTA alignment.

B. Multiple sequence alignment using CLUSTALW:

1. Open clustal omega.
2. Select FASTA alignment download file.
3. Run multiple sequence alignment.
4. Estimate evolutionary closeness of sequence by constructing phylogeny
tree.

Result: Acinetobacter lwoffii