PLNT2530 PLANT BIOTECHNOLOGY

Assignment 3 - March 17, 2017

This assignment is worth 5% of the course grade.

Due by 4:30 pm, Monday March 27

Inside your PLNT2530 directory, create a sub-directory called as3, to hold materials associated with Assignment 3. Save all files in your PLNT2530/as3 directory.

Copy the following files from your Ugene/Intro directory to your as3 directory:

pBS_SK+.gen
pBS_SK-.gen
pBS_SK-GUS.gb

1. (xx points) Demonstrate the effect of sequence topology on restriction digests.

It is critical to realize that the topology of a sequence ie. whether it is circular or linear, drastically affects the fragments predicted in a restriction digest. If the topology is set incorrectly for a sequence, the results of a restriction site search will be wrong.

save pBS_SK-GUS as a FASTA file
redo the BACHREST search
What are the differences between the digests for linear and circular sequences?

2. (xx points) Create a construct using pBluescript SK(+), to compare with the construct made using pBluescript SK(-).

Using the same procedures as detailed in the tutorial, create a construct by cloning the same 3 kb 35S-GUS fragment from pBI121 between the EcoRI and HindIII sites in pBluescript SK(+). Call this file pBS_SK+GUS.gb. For each construct, generate Export a map from the circular Overview in PNG format, using the file names pBS_SK+GUS.png and pBS_SK-GUS.png.

3. Use BACHREST to find restriction digests that would allow us to determine which of the two Bluescript vectors was used in the real construct. (xx points)

Read both pBS_SK-GUS.gb and pBS_SK+GUS.gb into bldna. Run BACHREST on both sequences to compare restriction fragments that would be generated by different enzymes. From the text editor, save the BACHREST output to pBS_SK-GUS.bachrest and pBS_SK+GUS.bachrest, respectively.

Comparison of the digests should reveal a number of enzymes that give easily distinguishable banding patterns if the real constructs were compared side by side on a gel. Because measurement of fragment sizes on gels is not very accurate, don't depend on being able to distinguish fragments whose sizes differ by < 10%. Also assume that measurement of fragment sizes below 200 bp will be even less precise.

In your report, display your results in a table, showing from both constructs side by side for comparison. You should be able to find at least three digests that easily distinguish between the two constructs.

As a second means of comparison, make two screenshots showing each vector in the Ugene Overview, with just the three restriction sites you have chosen.

Take screenshots of the following for your report:

4. (xx points)

The report should include the following:

Your name and student ID number.

Presentation guidelines

Screenshots should show the data requested, and be readable.
Screenshots should only show the relevant window(s), not the entire screen.
Blncbi screenshots for each query in your report should be presented as shown in examples.pdf.
Before taking a screenshot, resize the window, if possible, to minimize the size, while still retaining all necessary information.
When saving files in Linux, it is usually best NOT to include blank spaces in file names. If you need to separate words in a name, you can use other visible characters eg. bluescript_AND_syn.tsv.

Use fixed fonts for program output
Many programs generate output that only makes sense with a fixed font. Most fonts commonly-seen in documents are proportional fonts, meaning that narrow letters such as 'i' or 'l' take up very little width, whereas wide letters such as 'O' take up more space. In fixed fonts, all letter and numbers take up exactly the same width on a line of text. For example, output from the BACHREST program, listing restriction cutting sites and restriction fragments for a sequence cut with the enzyme AcoI, are shown in both fixed and proportional fonts. Make sure that when output is shown on a web page, it is in a fixed font.

fixed font # of
Enzyme Recognition Sequence Sites Sites Frags Begin End
------------------------------------------------------------------------------
AcoI Y^CCGGR 3
1289 2817 2533 5349
1556 1288 1 1288
2533 977 1556 2532
267 1289 1555

proportional font AcoI Y^CCGGR 3
1289 2817 2533 5349
1556 1288 1 1288
2533 977 1556 2532
267 1289 1555

Most fonts are proportional, eg. Helvetica, Times.
Examples of fixed fonts include Courier, Lucida typewriter and Terminal.

Submitting your assignment

Your PDF report, along with associated TSV and GenBank files, is due by 4:30 pm, Mon. March 27 on the PLNT2530 UMLearn dropbox site in the Assignment 3 folder. Files in word processing formats (.doc, .docx, .rtf, .odt) are not acceptable.

If you have questions, it may help to send me a message at frist@cc.umanitoba.ca.