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Microbiology Lab: Analyzing Cell Envelopes and Proteins using BLAST, Study notes of Biology

Faculté Universitaire des Sciences Agronomiques de GemblouxBiology

In this lab activity, students learn about the structure of gram positive and gram negative cell envelopes and use the basic local alignment search tool (blast) to analyze protein sequences. They identify the top blast hit, determine the protein function and organism origin, and apply this knowledge to determine if the organism is gram positive or negative.

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Microbiology I
BIOL211-CAD Lab
This activity introduces BLAST (Basic Local Alignment Search Tool), a valuable tool for analyzing
nucleic acid and protein sequence data. In addition, this activity highlights some important differences
between the cell envelopes of Gram positive and Gram negative bacteria.
A) Cell Envelope Review: Look at the diagrams below of Gram (+) and Gram (-) type cell
envelopes.
i) Label each cell envelope as being from a Gram (+) or Gram (-) cell type
ii) Label the components of each cell envelope using the list below
Note: not all cell-types have all the structures.
cytoplasmic membrane
outer membrane
membrane-bound proteins
peptidoglycan layer
periplasmic space
porins
pf2

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O utside cell Inside cell O ut side cell Inside ce ll Microbiology I BIOL211-CAD Lab This activity introduces BLAST (Basic Local Alignment Search Tool), a valuable tool for analyzing nucleic acid and protein sequence data. In addition, this activity highlights some important differences between the cell envelopes of Gram positive and Gram negative bacteria. A) Cell Envelope Review: Look at the diagrams below of Gram (+) and Gram (-) type cell envelopes. i) Label each cell envelope as being from a Gram (+) or Gram (-) cell type ii) Label the components of each cell envelope using the list below Note: not all cell-types have all the structures. cytoplasmic membrane outer membrane membrane-bound proteins peptidoglycan layer periplasmic space porins

Protein Organism % Que1y Coverage E-value % Max Identity Microbiology I BIOL211-CAD Lab B) BLAST review: One of the bioinformatic tools that you will use is BLAST ( Basic Local Alignment Search Tool ), that can be found at the National Center for Biotechnology Information site: http://blast.ncbi.nlm.nih.gov/. As the name implies, BLAST makes alignments between sequences. Alignment is the process (or result) of matching up the nucleotide or amino acid residues of two or more biological sequences to achieve the best possible match. BLAST identifies sequences similar to your query sequence in the NCBI database by making alignments and assessing how well the sequences match. Please view the BLAST Video Tutorial ( (https://www.youtube.com/watch?v=x_dAyY5-VNc) if you need additional help in answering the questions below. Below is the amino acid sequence of a protein associated with some bacterial cell envelopes. Use a protein BLAST ( BLASTP ) search to obtain information about it, and answer the questions below. MKLKNTLGVVIGSLVAASAMNAFAQGQNSVEIEAFGKRYFTDSVRNMKNADLYGGSIGYFLTD DVELALSYGEYHDVRGTYETGNKKVHGNLTSLDAIYHFGTPGVGLRPYVSAGLAHQNITNINSD SQGRQQMTMANIGAGLKYYFTENFFAKASLDGQYGLEKRDNGHQGEWMAGLGVGFNFGGSK AAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSK VKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGG RVNAVGYGESRPVADNATAEGRAINRRVEAEVEAEAK Questions: Identify the Top BLAST hit and fill in the box to answer questions 1-3.

  1. What kind of protein does this sequence encode, based on the name given (annotation)?
  2. From what organism did it come?
  3. What is the BLAST % query cover, E value and % Max Identity for the top hit? Top BLAST hit for the sequence from your isolate
  4. What is the function of this kind of protein?
  5. Based on what this protein does and where it is found, do you think this organism is a Gram positive or Gram negative bacterium? Explain your logic.
  6. Look at the BLAST tutorial (or look at the glossary section in the BLAST website at http://www.ncbi.nlm.nih.gov/books/NBK62051/) and fill in these definitions: E-value: % Max Identity: