Genetic engineering of pGLO plasmid

Bacterial cultures

http://www.cas.vanderbilt.edu/bsci111a/microbiology/supplemental.htm

Starch, amylase, and iodine test

1) Starch (amylose + amylopectin):

"Starch is generally insoluble in water at room temperature. Because of this, starch in nature is stored in cells as small granules which can be seen under a microscope. Starch granules are quite resistant to penetration by both water and hydrolytic enzymes due to the formation of hydrogen bonds within the same molecule and with other neighboring molecules.

However, these inter- and intra-HYDROGEN BONDS can become weak as the temperature of the suspension is raised. When an aqueous suspension of starch is heated, the hydrogen bonds break. "

(Source: http://www.eng.umd.edu/~nsw/ench485/lab5.htm)

Hence for boiled starch --> H bonds broken --> exposed amylose and amylopectin structure --> allow amylase to hydrolyse starch. 


In contrast, for unboiled starch --> amylase unable to hydrolyse starch as the starch granules are intact. 

2) Amylase:

Acts on α-1,4 glycosidic bonds (present in amylose and amylopectin). 

However, amylase is unable to break down amylopectin completely due to the α-1,6 glycosidic bonds present at the branch points. 

(Further reading: http://books.google.com.sg/books?id=xIj3YPKSpJkC&pg=PA453&lpg=PA453&dq=amylopectin+red+brown&source=bl&ots=uokPwYlqHY&sig=v_zrQ-kF7hCYj7So3lKARSbhBCo&hl=en&sa=X&ei=NM2dT6LCH8TJrAe8kcBm&ved=0CCcQ6AEwAQ#v=onepage&q=amylopectin%20red%20brown&f=false)

3) Iodine test 

The amylose (unbranched/linear portion of starch) forms helices, which allow iodine molecules to assemble, forming a dark blue/black color (refer to diagram below). 

The amylopectin (branched portion of starch) forms much shorter helices due to the branching present, and iodine molecules are unable to assemble, leading the color to be of red brown or red violet. 

As starch is broken down or hydrolyzed into smaller carbohydrate units, the blue-black color is not produced. Therefore, this test can determine completion of hydrolysis when a color change does not occur.

(Further reading: http://www.webexhibits.org/causesofcolor/6AC.html)

***Please refer to the SPA answer scheme (when it becomes available at IVLE) to see how the answers should be phrased for Q18,19 and 22.

How a light microscope works - FYI

http://www.microscopemaster.com/brightfield-microscopy.html

"In brightfield microscopy a specimen is placed on the stage of the microscope and incandescent light from the microscope’s light source is aimed at a lens beneath the specimen. This lens is called a condenser.

The condenser usually contains an aperture diaphragm to control and focus light on the specimen; light passes through the specimen and then is collected by an objective lens situated in a turret above the stage.

The objective magnifies the light and transmits it to an oracular lens or eyepiece and into the user’s eyes. Some of the light is absorbed by stains, pigmentation, or dense areas of the sample and this contrast allows you to see the specimen.

For good results with this microscopic technique, the microscope should have a light source that can provide intense illumination necessary at high magnifications and lower light levels for lower magnifications. "

Practical: Videos for use of a microscope

Using:
http://www.youtube.com/watch?v=X-w98KA8UqU&list=FL-cFTJ8NxBjonnjuwSQgSsw&index=10&feature=plpp_video

Focusing:
http://www.youtube.com/watch?v=scEhgAiazzU&feature=autoplay&list=FL-cFTJ8NxBjonnjuwSQgSsw&lf=plpp_video&playnext=1

Wet mount:
http://www.youtube.com/watch?v=jjevU-XMVzU&list=FL-cFTJ8NxBjonnjuwSQgSsw&index=13&feature=plpp_video

Graph drawing for lab: Sample of line of best fit

Line/curve of best fit best represents the data on a scatter plot. It may pass through none, some, or all of the points. Try to balance the number of points above and below the line.