MICR5831 L7: DNA Cloning II 8/5/25 NEEDS SAQ

What are the steps for screening for a gene of interest/to isolate a plasmid?

1) Screen for genes with the same phenotype

2) Pick a colony

3) Grow pure culture

4) Alkaline lysis or column purification of plasmid

5) Analyze plasmid sequence or via restriction map

How do you identify cloning vectors that successfully underwent recombination?

Insertional inactivation of phenotype present in plasmid

Name two examples of insertional inactivation of a phenotype expressed by the plasmid

1) Antibiotic resistance marker on PBR322

-Can use replica plating/picking and patching for recombinant vector

2) Blue white screening using pUC vectors

-Simple visual screen for recombinant vector

What is this?

-Tripartite replicon, first example of a modified vector

-Contains 3 segments:

-pMB1, R6-5, and R1

pBR322

What segments are found in pBR322, a tripartite replicon/the first modified vector?

1) pMB1:

-Segment from ColE1 plasmid

-oriV

-Medium copy number

2) R6-5:

-Segment from R plasmid

-Tetracycline resistance (TetR)

3) R1:

-Segment from R plasmid R1

-Ampicillin resistance (AmpR) from Tn3

What does this segment of PBR322 contribute?

pMB1:

-Segment from ColE1 plasmid

-Medium copy number

oriV (Origin of Replication)

What does this segment of PBR322 contribute?

R6-5:

-Segment from R plasmid

Tetracycline resistance (TetR)

What does this segment of PBR322 contribute?

-R1:

-Segment from R plasmid R1

Ampicillin resistance (AmpR) from Tn3

Name the steps for identifying recombinant vectors by using insertional activation (EX: pBR322)

1) Digest foreign DNA and pBR322 with SalI

2) Ligate digested DNA together

3) Transform into E. coli bacteria

4) Plate onto agar plates containing ampicillin

5) Replica plate colonies onto agar with only ampicillin or only tetracycline

What is this recombinant vector used for Blue-White Screening?

-Derived from pBR322

-MCS starts after lacZ gene

-Plac promoter for inducible expression

-VERY high copy number (~1000 copies/cell)

pUC series

How is pUC derived from pBR322?

-Replacing TetR-gene with lacZ' gene

-Gene for alpha-peptide of b-galactosidase

Where is the MCS (Multi-Cloning Site) of pUC found?

Starts after Nucleotide 15/Amino Acid 5 of the lacZ' gene

What does the Plac promoter of pUC do?

-Binds LacI repressor

-Induces transcription via allolactose, IPTG

True or False: Plac promoter of pUC can be turned on and off via inducible expression

True

Which produces more copies of genes, pUC or pBR322?

pUC

What does the B-galactosidase LacZ do?

-Cleaves lactose to galactose and glucose

-Cleaves X-gal into a blue precipitate

What peptides is LacZ cleaved into?

1) lacZ A M15

2) lacZ' alpha peptide

True or False: The peptides formed by LacZ being cleaved (lacZ A M15 and lacZ') are both inactive

True

True or False: Coexpression of both lacZ A M15 and lacZ' will restore ẞ-galactosidase activity via a-complementation

True

What is this?

-F' plasmid

-Contains lacZAM15 (inactive B-galactosidase)

-Contains lacl repressor

E. coli host JM109

What two genes does the JM109 E. coli host contain?

-lacI (repressor)

-lacZAM15 (inactive B-galactosidase)

True or False: The pUC vector's origin of replication is compatible with the F' one in JM109

True

Where can you find MCS (Multiple Cloning Sites)?

-pLac (promoter)

-lacZ' (inactive alpha peptide)

True or False: If the MCS contains a fragment of DNA, the complementation will be disrupted and appear as white (recombinant) instead of blue (wild type)

True

True or False: If 10 extra amino acids are added to lacZ', yet the translational frame is maintained and it can complement lacZAM15, it will not show up as recombinant (white) but rather the wild-type (blue-green)

True

What is the first step in Blue White screening for UC18?

1) Digest foreign DNA and pUC18 with Sal

What happens during Blue White screening after this?

1) Digest foreign DNA and pUC18 with Sal

Ligate digested DNA together

What happens during Blue White screening after this?

2) Ligate digested DNA together

Transform digested DNA into E. coli host JM109

What happens during Blue White screening after this?

3) Transform digested DNA into E. coli host JM109

Plate onto agar plates containing ampicillin, IPTG and X-gal

What happens during Blue White screening after this?

4) Plate onto agar plates containing ampicillin, IPTG and X-gal

White colonies contain recombinant vector

Blue/green colonies contain self-ligated vector

How do you screen a library for a specific gene?

1) Synthesize a labelled probe that binds nucleotide sequence of gene

2) Raise an antibody that binds protein

How do you design a nucleotide sequence probe?

1) Take homologous gene from a related organism

2) Purify protein, sequence and back-translate it to produce a degenerate nucleotide sequence probe (conserved motif with 90-100% identity)

How do you design a probe for a protein?

-Purification of protein encoded by the gene

-Raise antibodies that will detect protein expressed by the clones

How do you identify a particular gene using this method?

Create a hybrid colony with a nucleotide probe

1) Take individual white colonies

2) Place nitrocellulose filter on plate to pick up colonies

3) Incubate filter in alkaline solution to lyse bacterial cells and release the target DNA

4) Hybridize with labeled probe

5) Perform autoradiograph

6) Signal appears over colony that contains DNA which is complementary to the probe

How do you identify a particular gene using this method?

Immunoblot a colony with antibodies

1) Overlay nitrocellulose filter

2) break open bacteria with SDS and NaOH, then neutralize

3) Proteins from Master plate bind to nitrocellulose

4) Incubate filter with primary antibody then wash and incubate with radiolabeled secondary antibody

5) Fusion protein binds to nitrocellulose, primary antibody and secondary antibody bind to protein

6) Perform autoradiography via X-ray film to confirm protein's presence thanks to attached radiolabelled secondary antibody

Using a flow chart describe the steps you would use to isolate a gene of interest. (slide 27)

1) Screen

-Find Phenotype (ex: Haemolysis)

2) Pick colony

3) Grow a pure culture

4) Purify the plasmid

- Alkaline lysis method

-Column purification

4) Analyse plasmid

-Restriction map

-Sequence

Describe the process of insertional inactivation and how this can identify your gene of interest. (slide 30-31)

-Insertional inactivation of a phenotype expressed by the plasmid

-Antibiotic resistance marker on pBR322

-Replica plating/picking and patching for recombinant vector

-Blue white screening using pUC vectors

-Simple visual screen for recombinant vector

How does blue white screening work? (slide 34-35)

1. Plasmid Design:

   The plasmid vector used contains a multiple cloning site (MCS) within the lacZ gene. 

2. Insert Disruption:

   When a DNA insert is successfully ligated into the MCS, it disrupts the lacZ gene. 

3. Enzyme Production:

   If the lacZ gene is intact (as in non-recombinant plasmids), the bacteria will produce functional β-galactosidase. 

4. X-gal Conversion:

   β-galactosidase cleaves X-gal, producing a blue-colored compound. 

5. Colony Color:

   Colonies with intact lacZ (non-recombinant) appear blue, while those with a disrupted lacZ (recombinant) appear white. 

6. Selection and Screening:

   Blue-white screening is used in conjunction with antibiotic selection to ensure only cells with the plasmid grow, and then visually identify those with the insert. 

Name the compounds and their role in the media used for blue white screening (slide 34-35)

• X-gal:

  This colorless substrate is cleaved by β-galactosidase. The resulting product then spontaneously dimerizes and oxidizes to form a blue precipitate, causing colonies with functional β-galactosidase to appear blue. 

• IPTG:

  This molecule is an inducer of the lac operon, meaning it triggers the expression of the lacZ gene. In blue-white screening, IPTG ensures that the lacZ gene is transcribed, leading to the production of β-galactosidase. 

• β-galactosidase:

  This enzyme is encoded by the lacZ gene. It is crucial for the color change in blue-white screening. If the lacZ gene is disrupted by an inserted DNA fragment, β-galactosidase is not produced, and the colony remains white. 

What are the natural and synthetic substrates of beta-galactosidase. What are the products of the cleavage reaction be beta-galactosidase? (slide 34)

Products of Cleavage:

• Lactose (Natural)

  • Galactose

  • Glucose

• oNPG & pNPG (Synthetic)

  • o-nitrophenol (or p-nitrophenol)

  • Galactose 

What is the role of IPTG in blue white screening? (slide 34)

IPTG is an analog of galactose that induces the expression of lacZ gene. It should be noted that IPTG is not a substrate for β-galactosidase but only an inducer.

Using LacZ as an example describe alpha-complementation (slide 34)

Non-functional β-galactosidase enzyme is restored to functionality through the interaction of two separate polypeptide fragments.

lacZ A M15 and alpha peptide (lacZ') are inactive

Co-expression of both peptides in the same cell restores ẞ-galactosidase activity (a-complementation

Describe the features of the E. coli JM109 strain that makes it ideal for blue white screening (slide 35)

-F´ episome includes a functional lacZ gene (specifically lacZΔM15).

-This allows for alpha-complementation

Describe the features of the pUC vectors that make them ideal for blue white screening (slide 36)

Multiple cloning site (MCS) within the lacZ gene, which encodes for the α-peptide of β-galactosidase.

Describe colony immunoblotting with a nucleotide probe (slide 38)

1. 1. Colony Transfer:

   Colonies are transferred from the growth medium to a membrane (often nitrocellulose or nylon). This can be done by gently pressing the membrane onto the agar plate or by using a specialized colony transfer apparatus. 

2. 2. Cell Lysis:

   The transferred cells on the membrane are then lysed to release their DNA. This can be achieved by various methods, such as using chemicals or heat. 

3. 3. DNA Binding:

   The released DNA is then bound to the membrane, typically through a denaturation step that separates the double-stranded DNA into single strands. 

4. 4. Probe Hybridization:

   A labeled nucleotide probe, designed to be complementary to the target DNA sequence, is introduced to the membrane. The probe hybridizes (binds) to any complementary DNA sequences present on the membrane. 

5. 5. Detection:

   The probe is labeled with a detectable tag (e.g., radioactive isotope, fluorescent dye, or enzyme). After washing away unbound probe, the labeled probe that hybridized to the target DNA can be detected, indicating the presence of the target sequence in that specific colony. 

6. 6. Analysis:

   The location of the signal on the membrane corresponds to the location of the colonies containing the target DNA sequence. 

Describe colony immunoblotting with an antibody (slide 39)

SAQ

Describe the enzymatic activity of restriction endonucleases using specific examples

Restriction endonucleases, also known as restriction enzymes, are bacterial enzymes that cut DNA at specific nucleotide sequences, called restriction sites.

EcoRI: Recognizes GAATTC and cuts between the G and A on both strands, creating a 5' overhang.

HindIII: Recognizes AAGCTT and cuts between the A and A, also creating a 5' overhang.

SmaI: Cuts straight through both strands, producing blunt ends.

Calculate the frequency of cutting exhibited by restriction enzymes that recognize 4bp, 6bp, 8bp

With four possible bases (A, T, C, G), there are 4 possible base pairs for each position in the sequence. For a 4bp sequence, the probability of a specific sequence occurring is (1/4) (1/4) (1/4) * (1/4) = 1/256. Therefore, a 4bp recognition site is expected to occur once every 256 base pairs.

Similarly, for a 6bp sequence, the probability is (1/4)^6 = 1/4096. A 6bp recognition site is expected to occur once every 4096 base pairs.

For an 8bp sequence, the probability is (1/4)^8 = 1/65536. An 8bp recognition site is expected to occur once every 65,536 base pairs.

Explain DNA electrophoresis

DNA electrophoresis is a laboratory technique used to separate DNA fragments based on their size and electrical charge. It involves applying an electric field to force DNA molecules through a gel matrix, with smaller fragments moving faster and further than larger ones. This allows scientists to analyze, identify, and purify DNA fragments for various applications

Explain how DNA ligase or alkaline phosphatase or T4 DNA polymerase works (add diagram)

DNA ligase repairs broken DNA by forming a phosphodiester bond between a nearby 5' phosphate and 3' OH of the nicked or cut DNA strand.

In addition to duplex DNA, T4 DNA ligase can also seal single stranded cut in RNA or DNA/RNA hybrids.

Describe the methods of bacterial transformation

Bacterial transformation is a method of genetic engineering where bacteria take up foreign DNA from their environment.

There are two primary methods for inducing bacterial transformation: heat shock and electroporation.

In both methods, the key is to create temporary pores in the bacterial cell membrane, allowing the foreign DNA to enter.

Describe, using a diagram, the steps taken to clone gene.

Gene cloning involves isolating a gene of interest, inserting it into a vector (like a plasmid), introducing the vector into a host organism (like bacteria), and then selecting and growing the host cells that contain the desired gene.

1) Isolation

2) Selecting/cutting vector w/restriction enzyme

3) Ligation

4) Transformation (adding recombinant DNA to host)

5) Selection and screening of vector plasmids

6) Amplification and expression to confirm gene