BIOL130L Study Guide - Final Guide: Biuret Test, Paper Chromatography, Iodine Test

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blackgnat889

21 Apr 2014

School

University of Waterloo

Department

Biology

Course

BIOL130L

Professor

Dragana Miskovic

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Document Summary

Ensures that any changes in the experiment were as a result to the actual experimental results: these controls can be positive or negative. A positive control = designed/prepared in such a way that the sample. Starch is a mixture of 2 different polymers (amylose and amylopectin: amylose is an unbranched helical molecules with alpha 1,4 linkages, amylopectin is a branched straight molecule with alpha 1,6 linkages. Iodine solution reacts with the amylose to form the intense blue- black colour. Glycogen is similar to amylopectin but is more branched. Iodine solution reacts with glycogen"s multi-branched structure to form the less intense reddish-brown colour: negative result: substance remains yellow: The cupric ions of the benedict"s test will react with the free aldehyde groups at the end of the straight-chain sugar to reduce the ions into cu+ Also bear in mind, substances like fructose with free ketone group can also reduce benedict"s solution: positive result:

Related Questions

Benedicts Test for Reducing Sugars:

Tube Solution/food Color reaction to Benedicts solution Presence ofreducing sugar (green/reddish orange color)
1 1ml distilled water
2 good pinch glucose powder
3 good pinch table sugar (sucrose)
4 2 drops honey
5 finely chopped onion
6 finely chopped fruit
7 1ml milk
8
9
10

Which of the above solutions was included as your negative control?[1 pt]

Which of the above solutions was included as your positive control?[1 pt]

Which of the above foods/solutions tested positive for sugars? [4pts]

Explain why sucrose (table sugar) yielded the result above. [2pts]

What does this tell you about the way sugars are stored, forexample in onions? [2 pts]

Sugar is actually a common name for glucose. Glucose is stored asstarch in onions as it is a bulb present in plants.

Would you use the Benedicts test to investigate if a cake mix weresugar free? Explain your answer [2 pts]

The benedicts test cannot be used to determine if a cake mix issugar free or not. This is because benedicts test is used todetermine the presence of reducing sugars like maltose and lactose.Fructose also gives a positive test. We use sucrose in food so wecannot detect the presence or absence of sucrose in the cakemix

Iodine Test for Starch:

Depression Number Solution/food Color reaction to iodine solutionPresence of starch (bluish-black color)
1 Â½ ml distilled water
2 pinch glucose powder
3 pinch cornstarch
4 Â½ ml egg white
5 finely chopped onion
6 Finely chopped potato
7 1 drop honey
8
9
10

Which of the above solutions was included as your negative control?[1 pt]

Which of the above solutions was included as your positive control?[1 pt]

Which of the above foods/solutions tested positive for starch? [4pts]

Did any of the animal sources contain starch? Explain your answer.[2 pts]

Did any of the same foods test positive for both reducing sugars(Benedicts Test) and starch (Iodine Test)? Explain your answer. [4pts]

Proteins:
Tube Solution/food Color reaction to Biuret Reagent Presence ofprotein (violet color)
1 2ml distilled water
2 2ml milk
3 2ml egg white
4 2ml egg yolk
5 2ml honey
6
7
8
9
10

Which of the above foods/solutions tested positive for protein? [4pts]

Did any of the foods/solutions that tested positive for proteinalso test positive for reducing sugars (Benedicts Test) or starch(Iodine test)? Explain your answer. [2 pts]

How is the structure of proteins in plants and animals similar? [3pts]

How are proteins in plants and animals different? [3 pts]

Lipids:

Test Solution/food Grease spot indicating the presence oflipid
1 1ml distilled water
2 Oil-vinegar salad dressing
3 walnut
4 Egg white (wipe away excess)
5 Egg yolk (wipe away excess)
6
7
8
9
10

Which of the foods tested were positive for the presence of lipid?[4 pts]

Part 2 â Tracking Macromolecules in Your Diet

Include a copy of your ânutrient intakeâ and record the followingnutrient intakes in the space below. NOTE: Nutrient Intake is thename of the report generated at www.MyPyramid.gov [10 pts]

Kilocalories Percent Total Recommended
Energy Intake Intake

Carbohydrate ___________ ______________ 45-65%

Protein ________________ ______________ 10-35%

Total Fat _______________ _______________ 20-35%

Show your calculations in the space below

How did your consumption of macronutrients compare to therecommended intake?

orangehippopotamus244

BIOTECHNOLOGY

Introduction

In this Experiment we will learn how to identify the unknownbacterium according to the case study given as you start to performyour procedures and following the steps. Also, we will be learningthe outcome of a specific case scenario of immunity. We will beperforming different examples of media and observing the reactionafter inoculation and adding different reagents as it produce somechanging of color diagnose to a negative or positive results. Also,we will conduct different types of biochemical testing includingIndole production, Citrate Utilization, Methyl red test, andMannitol Fermentation.

In this case scenario we are trying to figure out theinfestation of food at the warehouse causing a death of theplaintiff mother. The plaintiff is accusing the warehouse forselling these infested products by insects. At the warehouseinspection is been done by swabbing and acquiring the type oforganism to be identified as evidence for the court finaldecision.

One of the major findings in this experiment is that itwas negative gram stains and the Indole and Methyl Red Test had apositive reaction. Additional testing was perform which show theresult of only negative on Lactose Fermentation and the remainingthree additional testing add up to a positive. The outcome of thisinvestigation scenario could be a negative result to the casescenario presented about infested products by insects since nounknown bacteria was left to bediagnosed.

Procedure

The following steps are requiring completing this labexperimental. First make sure you open the Virtual UnknownSoftware. Complete conducting using Indole production and CitrateUtilization, Methyl Red test choosing from your media option. Alsomake sure you open the reference book for each procedure to beinformed of the lab steps and decision making such as positive ornegative and its color after inoculation.

Observations and Results

	Result + or -	Appearance after Incubation
Gram Stain	- gram rods	N/A
Indole Production	+ positive	Yellowish-after incubation & adding Kovacâs reagent changingto a cherry red color indication of positive
Mannitol Fermentation	- neg	Red
Methyl Red Test	+ pos	Turbid yellowish after incubation and adding methyl red reagentchange to red positive. +
Citrate Utilization	- neg	Greenish after incubation. No reagent added.

Additional tests performed are listed below:

	Result + or -	Appearance after Incubation
Lactose Fermentation	- neg	Red- no reagent added
Sucrose Fermentation	+ pos	Yellowish- no reagent added
Phenol Red Rhamnose (Lactose Steps)	+ pos	Yellow- no reagent
Phenol Red Xylose (Sucrose Steps)	+ pos	Yellow- no reagent

Bacterium identified as: You may want to do some additionaltests to conclusively identify your bacterium. Some good ones to domight be lactose and sucrose fermentation, like you did last week.Remember, they use Phenol Red Lactose Broth and Phenol RedSucrose Broth and yellow= positiveand red =negative. After you do those two tests,you may want to do Phenol Red Rhamnose and Phenol RedXylose, which are set up the same way as the sucrose andlactose tests, they just have different sugars. Be sure to recordyour test results!

Discussion

Identify the bacterium â Make sure you look in the VirtualUnknown lab report to make sure you got the right organism. Afteryou have made your identification, look at the lab report. You cando this by going to the View menu in Virtual Unknown and selectingLab Report. You can double check with me that you are researchingthe right one.

Once all observation was performed including the additionaltesting I was left without an Unknown organism to be identified.This only leave me with a diagnose to state that no evidence wascollected to accused the warehouse as being infested with insectsaccording to the case study.

Explain what features of this organism make it an idealcandidate host for your insecticide. (Hint: remember you will needto infect insects with the bacteria to deliver the insecticide.)âFind out where the unknown organism was isolated from.

Describe in detail the steps you will take to insert the targetgene into the bacterium. - Figure 8.1 of your book should help youwith this.

Conclusion

< Within a few sentences, provide a concluding statementabout the results of your laboratory>

Virtual Lab Report

Student:

Instructor: Test User

Section:

Unknown: BiotechUnknown

Identification Information

Assigned Unknown: Identification not yet complete

Identified Unknown: TBD

Gram Reaction & Morphology: Gram Negative Rods

Auto-Inoculation Used: Yes

Test Detail (Chronological Order):

1 Gram Reaction {-} Eliminated {53}Remaining {71} Recorded 1 time(s)

Observations: none

2 Indole production {+} Eliminated {44}Remaining {27} Recorded 1 time(s)

Observations: none

3 Mannitol fermentation {-} Eliminated {19}Remaining {8} Recorded 1 time(s)

Observations: none

4 Methyl red {+} Eliminated {0} Remaining{8} Recorded 1 time(s)

Observations: none

5 Citrate Utilization (Simmons) {-}Eliminated {2} Remaining {6} Recorded 1 time(s)

Observations: none

6 Lactose fermentation {-} Eliminated {0}Remaining {6} Recorded 1 time(s)

Observations: none

7 Sucrose fermentation {+} Eliminated {4}Remaining {2} Recorded 1 time(s)

Observations: none

8 Rhamnose fermentation {+} Eliminated {2}Remaining {0} Recorded 1 time(s)

Observations: none

9 Xylose fermentation {+} Eliminated {0}Remaining {0} Recorded

goldturtle749

The overall goal of the research project that we carried out in Experimental Biology is insertion of GFP into target lapA/EF-4 gene.

1. What is the overall goal of the research project that we carried out in Experimental Biology, and how does the experiment aimed at purification of your fusion protein fit in with the overall goal of our research? How does the subcellular localization experiment fit into the overall goal?

2. What method did we use to determine whether your fusion protein was a soluble protein or a membrane bound protein? What positive control did we perform? What result was obtained from the positive control? Was this expected? Explain.

3. Know the methods used for protein purification in general as described in class, and in the file; âprotein purification.pptâ

4. What method did we use to attempt to purify your LepA/EF 4 â GFP fusion protein? Why did we use this method, what advantages are there compared to other protein purification methods?

5. What is âProtein Aâ and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why canât we use the anti-EF 4/LepA antibody to purify your fusion protein?

DAY 18, NOVEMBER 7: INTRODUCTION TO IN VIVO FUNCTIONAL ASSAYS; ASSAY FOR EXPRESSION TOXICITY AT 30Â°C VS. 37Â°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-FâlacI^Q -/+ IPTG to compare toxicity of functional LepA/EF-4 (expressed from pPEM109) to empty vector control (pIN III), and EF-4-GFP fusion proteins.

Background: This assay is based on the observation that E. coli is extremely sensitive to elevated levels of LepA/EF-4 expression. In prior work it was demonstrated that LepA/EF-4 expression from pPEM109 is lethal to cells growing in the presence of IPTG [Yaskowiak and March, 1995). Furthermore, pPEM109 can only be maintained in strains that contain the mutated Lac repressor gene, lacI^Q, which increases repression of the operon. The wild-type promoter of the lacI gene is rather weak, resulting in ~10 molecules of Lac repressor protein per wild-type cell. The lacI^Q gene contains a âpromoter-upâ mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (MÃ¼ller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of âleakyâ expression from the lac promoter. It was shown by comparing two identical strains (one contained lacI^Q and one contained lacI) that pPEM109 could only be maintained in the lacI^Q containing strain.

If you consider the structure of LepA/EF-4, and its proposed function, why do you think it would be toxic if expressed at high levels levels? What kind of mutations in the lepA gene could give rise to a mutated protein that is not toxic when expressed? What do you predict for your LepA/EF-4 - GFP fusion protein, will it be toxic when expressed or not?

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DAY 19, NOVEMBER 9: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Lysis and Ultracentrifugation).

Background: LepA/EF-4 was originally found in the cytoplasm, membrane and periplasm when the protein was expressed from a plasmid [March and Inouye (1985) J. Biol. Chem. 260: 7206-7213]. The endogenous protein, expressed from the chromosomal gene in the absence of plasmid expression was located in the membrane [March and Inouye (1985) Proc Natl Acad Sci, USA Vol. 82: 7500-7504]. More recently it has been shown that the cellular location of LepA/EF-4 depends on the Mg²⁺ stress status of the cell [Pech et al. (2011) Proc Natl Acad Sci, USA Vol. 108: 3199-3203].

Here we will examine the comparative distribution of LepA/EF-4 and LepA/EF-4-GFP in cells by subcellular fractionation of native cellular protein extracts. Do you predict that your EF-4-GFP fusion protein will be localized to the cytoplasm, membrane, or both?

In contrast to our previous denaturing extract preparation, the native preparation maintains the overall shape/folding of proteins so they can localize appropriately prior to analysis. Looking ahead, how will the preparation be different in native vs. denaturing extracts?

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DAY 20, NOVEMBER 14: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (SDS PAGE and Transfer)

Procedure:

SDS PAGE Analysis

1. Each section should have the following control samples to analyze from DAY 19:

Extract Fraction Treatment

pPEM109 Input +IPTG

pPEM109 Sol +IPTG

pPEM109 Mem +IPTG

In addition, each student should have 3 experimental samples for their candidate:

Extract Fraction Treatment

Candidate Input +IPTG

Candidate Sol +IPTG

Candidate Mem +IPTG

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DAY 21, NOVEMBER 16: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Western Blot detection)

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DAY 23, NOVEMBER 28: PURIFICATION OF NATIVE LEPA/EF 4 â GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 â GFP fusion proteins retain function our prior experiments examined whether fusion proteins retained expression toxicity, and whether fusion proteins localized to the membrane. In order to examine other aspects of function, such as GTP â binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 â GFP fusion proteins using affinity purification.

Background: Scientists are often interested in recovering proteins that have been produced in bacteria for research or therapeutic purposes. The process of protein purification from bacteria is medically relevant. It can be used as a method to mass-produce proteins of interest. For instance, the human insulin gene can be cloned into a plasmid and transformed into E. coli. The protein can then be purified and used to treat patients with diabetes.

The process of purifying a protein means separating this protein from other cellular components (proteins, DNA, ions, etc.). Protein purification is described in all biochemistry books, and in Chapter 5, Section A, of the textbook used in the Emmanuel College Biochemistry class (Fundamentals of Biochemistry, Voet, Voet and Pratt). This book is on reserve in the Emmanuel College Library. Here we will test the feasibility of using affinity chromatography, and magnetic beads to purify LepA/EF 4 fusion proteins in one step.

Preparation of magnetic beads for affinity purification of native LepA/EF 4. This procedure will be done the day before by the Emmanuel College Lab staff.

Background. The primary antibody that we employed to detect LepA/EF 4 â GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 â GFP fusion protein. Commercially available Protein A magnetic beads will be used for this purpose. Protein A is a very stable bacterial protein that has a high affinity for IgG produced from any animal species. Protein A magnetic beads contain tiny magnetized beads with Protein A covalently attached to their surface. Remember that the primary antibody that was employed in our prior experiments was affinity-purified rabbit IgG that was specific for GFP epitopes. The application of GFP primary antibody to Protein A magnetic beads will result in a tight, non-covalent attachment of GFP â specific IgG onto the magnetic beads. These beads would specifically recognize and bind to GFP that was present in a crude cell lysate. The application of a magnetic field could then be employed to collect the beads and the bound LepA/EF 4. By this method the collection and wash steps would be very rapid and very efficient. After washing away unbound proteins, purified LepA/EF 4 â GFP could be eluted from the magnetic beads. HOWEVER, the GFP IgG was non-covalently attached to the Protein A, and therefore some will leak out in the elution step. This would contaminate the purified LepA/EF 4 â GFP fusion protein with IgG. To avoid this problem, GFP IgG will be chemically cross-linked onto the Protein A magnetic beads by the procedure described in Sisson and Castor [Journal of Immunological Methods (1990), Vol. 127, 215-220].

BIOL130L Study Guide - Final Guide: Biuret Test, Paper Chromatography, Iodine Test

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