Applied Molecular Biology

Applied Molecular Biology MBIO 4672 Assignment

The term assignment consists of starting with a DNA sequence (contig) that was generated by a recent pyrosequencing run of R. leguminosarum strain VF39 and using online tools to identify and annotate the open reading frames found on the contig that has been assigned to you. The contigs that have been selected are approximiately 10 kb.  They have not been cleaned up in any way-expect to have partial orfs at the beginning and end of each contig.  In addition to identifying the orfs, you will be have to focus on a single orf from your contig.  Using results from your initial bioinformatic characterization, the remainder of the assignment consists of designing a logical experiment(s) that can be used to test the hypotheses generated from your initial characterization. Each person has been given a personalized sequence with which to work. 

Due Date:  Assignments should be uploaded onto D2L site. The dropbox is set up to take upload files until 12 PM Friday April 10, 2015. The initial GeneMark output, a graphic showing the ORFs in addition to the annotation notebooks should be combined as one file, converted to a single pdf file and uploaded.

Accessing Sequence Data:  Sequence data can be accessed at this site http://home.cc.umanitoba.ca/~oresniki/lab_web_page/Sequence.html Click on the appropriate contig.  Save the sequence as a .txt file. 

>contig00055  genome.gb, 812490..823068    

Identification of ORFs (5 marks).  This section will include the initial identification of the ORFs found on the contig.  The results should be presented as a graphic.  Any other relevant information to support your hypothesis should be included (top BLAST hits, probability of reading frame, choice of start codons etc).

Useful sites

NCBI:                                                  http://www.ncbi.nlm.nih.gov/

GeneMark.hmm                      http://opal.biology.gatech.edu/GeneMark/

ORF annotation (5 marks)

When the orfs have been identified using GeneMark, use the gene annotation notebook to gather information information to help annotate the product encoded by 5 comnplete genes.  In some cases, you may find that best result may be a hypothetical protein that is conserved in a number of other bacteria.  In other cases, biochemical function may be assignable. If a gene has been previously characterized, provide the reference in addition to the BLAST results as evidence.

Useful sites

BLASTP/CD                          http://blast.ncbi.nlm.nih.gov/

ABIM online analysis tools    http://sites.univ-provence.fr/~wabim/english/logligne.html

Functional charactirization (10 marks).  In this section you will choose one of the orfs on your contig, provide logic and rationale why it was chosen, and design initial experiments that may lead to a experimental evidence with which to be able to assign functionality to an ORF. In this section you will need to include any primers and experimental details necessary to test the hypotheses you have generated in the previous section.  Please note that many of the materials and methods have been previously established and published.  Vectors or resources that are used to carry out the experimental portion need to be referenced.

Useful site for restriction analysis.

http://bio.lundberg.gu.se/cutter2/

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