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30 Jan 2019

1.In the T7/lac expression system used for ADH expression, which of the following is true?

The promoter for the ADH gene is specific for E. coli RNA polymerase.

The promoter for the ADH gene contains the lac operator sequence.

The pLacI plasmid encodes the T7 RNA polymerase gene.

All of the above

2.The ADH gene used in this lab contains

A his-tag at the N-terminus

An HSV tag near the N-terminus

A strep tag at the C-terminus

None of the above

3.During ADH expression in Tuner cells, which of the following occurs?

Binding of IPTG to the Lac repressor protein facilitates expression of the T7 RNA polymerase.

IPTG represses the expression of the Lac repressor protein.

The presence of glucose in the medium results in CAP-binding protein recruiting E. coli RNA polymerase to the T7 RNA polymerase gene.

All of the above

4. How would you interpret your NovaBlue transformation results if you saw no colonies on any of your plates (controls and experimental)?

You used the wrong cells for the transformation.

You used the wrong medium for the transformation.

You heat shocked the cells for too long.

Any of the above could explain the results.

5.In SDS-PAGE, which of the following is true?

The proteins are only negatively charged in the resolving gel.

The stacking gel has a higher pH than that of the resolving gel.

The concentration of acrylamide is higher in the stacking gel relative to the resolving gel.

none of the above

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Casey DurganLv2

31 Jan 2019

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You will be given two different plasmids, pLIT-GFP andpET28-bgal (Figure 2). pLITGFP has an ampicillin resistance (ampR)gene and pET28b-gal has a kanamycin resistance (kanR) gene. Youwill cleave both plasmids with restriction enzymes NcoI and XhoI,to release the fragment encoding green fluorescent protein (GFP)from pLITGFP, and ligate it with the vector pET28 cleaved with thesame enzymes. E. coli strain BL21(DE3) cells are transformed withthe recombinant plasmid mixture. Selecting for cells that containthe desired recombinant plasmid, pET-GFP, is done by platingtransformants on LB-agar that contains 50 Âµg/ml of kanamycin. Asantibiotics are heat sensitive, a sterile solution with theantibiotics is added to the agar after autoclaving, when the agarhas cooled down to about 55Â°C. pLIT-GFP (AmpR, ampicillinresistance marker) contains the GFP coding sequence between NcoIand XhoI sites. Ligation of the NcoI-XhoI digests of pLIT-GFP andpET28-bgal (KanR, kanamycin resistance marker) yields severaldifferent products, one of which is pET28-GFP, in which GFP gene issubstituted for beta-galactosidase. The T7 promoter drivestranscription of the adjacent gene when T7 RNA polymerase ispresent.

Considering the components of the ligation reaction forExperiment 1, Week 1, list all of the possible ligation productsthat could occur. Which of these products will confer kanamycinresistance in transformed bacteria?

copperseal477

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].

QUESTION 10

If you can drink milk as an adult, it means that you have inherited a mutation in the promoter of your lactase gene (the gene that encodes the enzyme you need to break down lactose). Predict the effect of this mutation:

		The mutation changes the number of domains in the enzyme, which makes it work more efficiently
		The mutation changes the amino acid sequence of the lactase protein
		The mutation increases the number of copies of the lactase gene that will be found in your genome
		The mutation changes whether the lactase sequence is found in an intron or exon
		The mutation affects the expression of the lactase gene

1.2 points

QUESTION 11

A competitive inhibitor is decreasing the activity of an enzyme. Predict the effect of adding more substrate to the reaction.

		The substrate will increase the reaction rate by binding to the allosteric site
		The substrate will increase the reaction rate by competing with the inhibitor for the active site
		The reaction rate will not change unless the inhibitor can be removed
		The enzyme adjusts its shape so that the substrate, but NOT the competitive inhibitor, can bind
		The substrate will bind to the competitive inhibitor and block its ability to bind to the enzyme

1.2 points

QUESTION 12

What determines where in the genome a transcription regulator will bind?

		Transcription regulators bind to the 5' UTR region of a gene
		Regulators bind via complementary base-pairing to certain DNA molecules
		Covalent bonds form between the transcription regulator and the atoms of the DNA backbone
		Every eukaryotic gene has a different transcription regulator that will bind to the 5' end of the gene
		Transcription regulators bind to specific DNA sequences via multiple weak non-covalent interactions

1.2 points

QUESTION 13

What is the basic premise of cell theory?

		DNA -> RNA -> protein
		All cells arise from pre-existing cells
		DNA provides the complete instructions to create a cell
		The identity of a cell is determined through gene expression patterns
		All cells contain the same four basic macromolecules

1.2 points

QUESTION 14

What is the benefit of using BOTH the lac activator and the lac repressor to control gene expression?

		Using both an activator and repressor enables cells to more accurately determine the amount of lactose available in the environment
		Enzymes to digest lactose are only made when energy is low and lactose is available
		The activator can override the inhibition of the lac operon by the repressor
		The repressor can control the enhancer, while the activator can control the promoter
		When neither the lac activator or repressor is present, expression of the lac operon is too high

1.2 points

QUESTION 15

What is the histone code used for?

		Phosphorylation and acetylation of DNA affect its ability to be compacted
		Changes to the sequence of DNA change whether DNA will wrap around histone proteins
		Covalent modifications of histones affect the ability of the transcription initiation complex to form
		Histones provide the codon sequences needed for translation to occur
		The histone code affects which amino acids will get added to a polypeptide