🏷️ LIMITED TIME OFFER: GET 20% OFF GRADE+ YEARLY SUBSCRIPTION →
Log in
Biology
1
answer
0
watching
108
views

Suppose that recombinant DNA technology was used to make a gene in which the structural genes of the lac operon were placed immediately downstream of the promoter and operator of the trp operon. This new operon is then introduced into the chromosome of an E. coli strain that does not have the normal lac operon. Therefore, the only way to get expression of β-galactosidase is from this artificial gene.

Bacteria with this artificial gene were grown under various conditions, and the level of β-galactosidase expression was measured. The effect of mutation of the trpR gene was also evaluated. The results from these experiments are given below:

presence of

tryptophan

presence of

glucose

presence of lactose

genotype

Level of β-galactosidase

expression:

present

absent

present

trpR+ trpO+

low

absent

present

present

trpR+ trpO+

high

present

absent

present

trpR- trpO+

high

absent

present

present

trpR- trpO+

high

Can someone please explain to me in detail why the levels of β-galactosidase happened to turn out like this?

Watch
1
answer
0
watching
108
views

For unlimited access to Homework Help, a Homework+ subscription is required.

Bunny Greenfelder
Bunny GreenfelderLv2
28 Sep 2019

Unlock all answers

Get 1 free homework help answer.
Already have an account? Log in
Share

Related textbook solutions

Molecular Cell Biology
8 Edition,
Lodish
ISBN: 9781464183393
Biology: Science for Life with Physiology
6 Edition,
Belk
ISBN: 9780134555430
Concepts of Biology
OER Edition,
Openstax
ISBN: 9781938168116
Biology
2nd Edition, 2013
Openstax
ISBN: 9781947172517
Essentials of Biology
5 Edition,
Mader
ISBN: 9781259660269

Related questions

Are my answers correct?

1. If a bacterium with a wildtype lac operon is grown in the presence of lactose, high levels of transcription result for a while, however, eventually transcription is again down-regulated. If, on the other hand, the sugar IPTG is present (instead of lactose), transcription levels are high and remain high. What function do IPTG and lactose share? How are they differently affected by the operon product?

Lactose and IPTG are both inducers of the Lac-Operon. Lactose is a substance that the enzyme will react with (substrate), which is split or cleaved by the Beta-galactosidase. When you add lactose expressions level of lac operon will increase but as lactose is used by the beta-galactosidase expression levels of lac operon will decrease.

IPTG is a chemical cannot be split or cleaved by beta-galactosidase. The expression level of lac operon will remain the same.

Or Lactose can be easily broken down but IPTG cannot be broken down. What does expressions level mean?

2. If a lacI - bacterium is grown in the presence of the sugar X-gal, a blue dye appears; however, the concentration of X-gal decreases over time. What has happened to the X-gal?


If a lacI - bacterium is grown in the presence of the sugar X-gal, the Lacl is mutant or the gene is damaged which alters the genetic message carried by that gene. The LacI gene produces the repressor protein which switches off the operon, but since it is damaged no repressor protein is produced and the operon is switched on. The Lacl is transcribed into beta-galactosidase (lactose) The enzyme beta-galactosidase (lactose) splits or separates the X-gal which will result in decreased concentration of X-gal with time.

3. If a wildtype bacterium is grown in the presence of the sugar X-gal, transcription of the polycistronic m-RNA does not occur. What is X-gal unable to do? Without adding lactose (or genetically modifying the bacteria), how could you fix this problem

X-gal cannot form lactose, the repressor remains off and beta-galactosidase is not formed. This can be fixed by adding IPTG which can switch on the operon

For this question, focus on four parts:

I, the gene that produces the repressor protein;

P, the promoter region where RNA polymerase binds to initiate transcription;

O, the Operator site, where the repressor binds. When the repressor binds, it prevents transcription; and

ZYA, the three structural genes, all of which are required for the successful utilization of lactose as a food source.

In the left column of the table below, we indicate the genetic composition at the lac operon of particular strains of E. coli cells. A "+" (plus sign) following the letter indicates the wild-type allele (or normally functioning version of the gene). For I, P, and ZYA, a "-" (negative sign) following the letter indicates the mutant allele (or non-functional version of the gene). For the O allele, a "C" indicates the mutant or constitutive allele. It is not functional in that the repressor cannot bind, but it is functional in that transcription can still occur.

Given the conditions stated in each column, please indicate for each genotype whether lac mRNA is produced and at what level by typing in one of the following words in each cell in the table:

"Abundant"

"Minimal"

"None"

The completely wild-type genotype has already been filled in.

Cellular conditions:
Genotype Glucose present (low cAMP) and no lactose Glucose present (low cAMP) and lactose present No glucose (high cAMP) and lactose present
I+ P+ O+ ZYA+ None Minimal Abundant
I- P+ O+ ZYA+
I+ P- O+ ZYA+
I+ P+ Oc ZYA+
I+ P+ O+ ZYA-

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; “protein purification.ppt”

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

5. What is “Protein A” and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why can’t 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 30°C VS. 37°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-F’lacIQ -/+ 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, lacIQ, 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 lacIQ gene contains a “promoter-up” mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (Müller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of “leaky” expression from the lac promoter. It was shown by comparing two identical strains (one contained lacIQ and one contained lacI) that pPEM109 could only be maintained in the lacIQ 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?

-------------------------------------------------------------------------------------------------------------------

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?

-------------------------------------------------------------------------------------------------------------------

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

-------------------------------------------------------------------------------------------------------------------

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

-------------------------------------------------------------------------------------------------------------------

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

Purpose: In order to examine whether LepA/EF 4 – 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 – binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 – 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 – GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 – 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 – 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 – 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 – 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].

Weekly leaderboard

Home
Homework Help 3,900,000
Biology 460,000
Start filling in the gaps now
Log in