Activity 2: Bacterial Transformation

Question 1

the process of making cells/organisms acquire/take up and express a new piece of foreign genetic material/DNA

Answer 1

Genetic transformation / Recombinant DNA Technology / Molecular or DNA Cloning / Genetic Engineering

Question 2

Typically, the goal in performing _____________ is to force the cell to express/produce the protein that the newly introduced foreign DNA codes for protein-coding gene

Answer 2

genetic transformation or genetic engineering

Question 3

Central dogma of biology

Answer 3

REPLICATION
TRANSCRIPTION
TRANSLATION

Question 4

1. Identify a gene for a protein.
2. Put the gene into bacteria.
3. Grow lots of the bacteria.
4. The bacteria transcribe and translate the gene - mini protein factories!
5. Purify the protein of interest.

Answer 4

Overview of transformation: How can we produce the protein of interest?

Question 5

Genetic engineering using

Answer 5

plasmids

Question 6

______ often have plasmids - extrachromosomal circular loops of DNA

can take in new plasmids

Answer 6

Bacteria

Question 7

plasmid artificially inserted with foreign DNA segment, such as a foreign protein-coding gene

Answer 7

Recombinant plasmid

Question 8

is the preferred term for the technique to artificially introduce recombinant plasmid to bacterial and plant cells

Answer 8

“Transformation”

Question 9

-PGLO LB
-PGLO LB/amp
+pGLO LB/amp
+PGLO LB/amp/ara

Answer 9

proliferates with ecoli only
no growth
with growth
with growth and glow

Question 10

Which of the four culture plates should have green-colored bacterial colonies under UV light?

Answer 10

+PGLO LB/amp/ara

Question 11

One of three types of horizontal/lateral gene transfer that naturally occurs in bacteria

Answer 11

Transformation

Question 12

Natural process in which an organism transfers genetic material to another organism that is not its offspring.

Answer 12

Horizontal/Lateral Gene Transfer

Question 13

3 types of horizontal gene transfer

Answer 13

Transformation
Transduction
conjugation

Question 14

3 types of horizontal gene transfer
the uptake of exogenous DNA from the surrounding environment.

Answer 14

Transformation

Question 15

3 types of horizontal gene transfer
the transfer of DNA from one bacterium to another via cell-to-cell contact.

Answer 15

Conjugation

Question 16

3 types of horizontal gene transfer
DNA transfer occurs when the bacteriophage (virus) infects a donor bacterium and transports parts of that bacteria’s DNA into a new recipient bacterial
cell.

Answer 16

Transduction

Question 17

TRANSDUCTION phases

Answer 17

Infection
Lyse
Dissemination
Recombinaison homologue

Question 18

Foreign DNA introduced into an organism

Answer 18

Transgene

Question 19

Plasmid artificially inserted with foreign DNA segment

Answer 19

Recombinant plasmid

Question 20

DNA molecule used as a vehicle to carry a particular DNA segment (the foreign DNA) into a host cell (E.g., plasmid, virus DNA)

Answer 20

Vector/Construct

Question 21

Protein produced by the inserted foreign DNA

Answer 21

Recombinant protein

Question 22

Unicellular or multicellular organism whose genome has been artificially altered by the introduction of one or more foreign DNA sequences from another organism; “genetically modified organism”

Answer 22

Transgenic

Question 23

Artificial methods involving the introduction of foreign DNA in viral vector to prokaryotic or eukaryotic cell

Answer 23

Transduction

Question 24

Various artificial means of introducing recombinant plasmid to animal
eukaryotic cells

Answer 24

Transfection

Question 25

is the preferred term for the technique to artificially introduce recombinant plasmid to bacterial and plant cells

Answer 25

Transformation

Question 26

Cell that has taken up the recombinant plasmid and has acquired a new trait or phenotype after expressing the transgene

Answer 26

Transformed cell

Question 27

In our activity, the plasmid we will use is called was inserted with the jellyfish gene for green fluorescent protein

Answer 27

pGLO plasmid

Question 28

what jellyfish has a gene for green fluorescent protein which glows green under UV ligh?

Answer 28

Aequorea victoria

Question 29

protein that glow green under UV light Originally Isolated from the jellyfish Aequorea victoria

Answer 29

Green fluorescent protein (GFP)

Question 30

Is GFP Naturally occurring in many bioluminescent jelly fish, reef corals and marine crustaceans?

Answer 30

true

Question 31

Recombinant GFP has _______ amino acids

Answer 31

239

Question 32

Recombinant GFP has __________ Dalton (Da) protein

Answer 32

26,870

Question 33

what is the structure of a GFP?

Answer 33

Barrel structure with the fluorescent chromophore at center of the protein

Question 34

is GFP a "Nobel prize-winning" molecule?

Answer 34

true

Question 35

What is a chromophore?

Answer 35

A group of atoms forming part of an organic molecule that causes it to appear colored

Question 36

The GFP chromophore is comprised of ___________ adjacent amino acids.

Answer 36

three

Question 37

These amino acids in the GFP are enzymatically converted to an

Answer 37

active cyclic chromophore

Question 38

Using GFP as a biological tracer or reporter protein

Answer 38

true

Question 39

GFP Absorbs at nm Emits at nm

Answer 39

Absorbs at 395 nm Emits at 509 nm

Question 40

In vitro, _________ is used to excite the GFP chromophore, absorbing light at a wavelength of 395 nm, and emitting at a longer wavelength of 509 nm visible fluorescent green light

Answer 40

UV light

Question 41

GFP is a visual marker

Answer 41

true

Question 42

how does GFP work as a visual marker?

Answer 42

- Study of biological processes (example: synthesis of proteins) - Localization and regulation of gene expression - Cell movement -Cell fate during development - Formation of different organs - Screenable marker to identify transgenic organisms

Question 43

also known as a polylinker, is a short segment of DNA within a cloning vector (like a plasmid) that contains multiple unique restriction enzyme recognition sites.

Answer 43

multiple cloning site (MCS)

Question 44

Plasmid containing GFP gene fused to ____________ was inserted into cultured mammalian cells

Answer 44

Nup98 gene

Question 45

Scientists can modify or engineer plasmids for specific purposes.

Answer 45

true

Question 46

gene in pGLO plasmid that Allows transformed bacteria to survive on plates with antibiotic

Answer 46

Antibiotic resistance gene

Question 47

Genes for producing the desired protein or trait/phenotype for example, the gfp gene in the PGLO plasmid

Answer 47

Gene/s of interest

Question 48

The PGLO plasmid is engineered to have the GFP gene from

Answer 48

Aequorea victoria

Question 49

→ 3 genes present in the pGLO plasmid:

Answer 49

GFP, bla, and araC

Question 50

→ 3 genes present in the pGLO plasmid: Allows transformed bacteria to survive on plates with ampicillin.

Answer 50

Beta-lactamase (bla)

Question 51

→ 3 genes present in the pGLO plasmid: Gene for the protein _________ that controls the GFP gene like and ON/OFF switch.

Answer 51

AraC

Question 52

→ 3 genes present in the pGLO plasmid: Gene for green fluorescent protein.

Answer 52

GFP

Question 53

what is the selectable marker in pGLO? and how?

Answer 53

The selectable marker in the pGLO plasmid is the ampicillin resistance gene (bla). This gene encodes the enzyme beta-lactamase, which breaks down the antibiotic ampicillin. When bacteria are transformed with the pGLO plasmid, they become resistant to ampicillin, allowing scientists to select for the transformed cells by growing them on a medium containing ampicillin.

Question 54

Components of the pGLO plasmid Origin of replication; DNA sequence where replication of the plasmid begins (Without the ori, the cell cannot make copies of the plasmid)

Answer 54

ori

Question 55

Components of the pGLO plasmid The antibiotic resistance gene (Selectable marker); gene that encodes B- lactamase, an enzyme that breaks down the antibiotic ampicillin;

Answer 55

bla

Question 56

transformants expressing the ________ gene can be selected by placing ampicillin in the growth medium

Answer 56

bla

Question 57

Components of the pGLO plasmid A region containing restriction sites (e.g., Ndel, Hindlil, EcoRI, etc.), sequences that permit the insertion of foreign DNA in the plasmid

Answer 57

Multiple cloning site (MCS)

Question 58

Components of the pGLO plasmid Jellyfish gene that encodes green fluorescent protein; the foreign DNA (gene) inserted in the PGLO plasmid

Answer 58

GFP

Question 59

Components of the pGLO plasmid Promoter sequence for the inserted GFP gene

Answer 59

PBAD promoter

Question 60

Without arabinose in the growth medium, what protein blocks this sequence, and RNA polymerase cannot bind and initiate transcription of the GFP gene (No GFP protein produced)

Answer 60

araC

Question 61

Components of the pGLO plasmid Gene that encodes the regulatory protein that prevents RNA polymerase from binding to the PBAD promoter in the absence of arabinose

Answer 61

araC

Question 62

Only when __________ is present and binds to the araC protein will the production of GFP protein be switched on

Answer 62

arabinose

Question 63

gfp, araC, and bla gene in the pGLO plasmid have different promoter sequences; only the ___________ for gfp is usually shown in the diagrams of the pGLO plasmid

Answer 63

promoter (pBAD)

Question 64

The foreign DNA and the plasmid _____________should be cut with the same so that the foreign DNA can be inserted in the plasmid

Answer 64

restriction enzyme

Question 65

o Restriction enzymes differ in the DNA sequences that they can cut

Answer 65

true

Question 66

what connects the backbone of the foreign DNA to the backbone of the plasmid?

Answer 66

DNA ligase

Question 67

PGLO Plasmid: inserting the transgene (gfp) → araB, araA, & araD genes replaced with the _______

Answer 67

gfp gene

Question 68

set of related genes transcribed from the same promoter; common in bacteria

Answer 68

Operon

Question 69

In the absence of the sugar ________, araC protein causes DNA folding which prevents RNA polymerase from binding to the PBAD promoter → araBAD genes (replaced by gfp gene in the pGLO plasmid) cannot be transcribed

Answer 69

arabinose

Question 70

why do you add the cacl2 transformation solution?

Answer 70

In bacterial transformation, calcium chloride (CaCl2) is added to make bacterial cells competent, meaning they are able to take up foreign DNA. The CaCl2 helps neutralize the negative charges on the bacterial cell membrane, allowing the negatively charged DNA to more easily enter the cell. This process, combined with a heat shock, facilitates the uptake of DNA into the bacterial cell.

Question 71

________ genes are always expressed (e.g., araC gene, bla gene)

Answer 71

* Constitutive

Question 72

genes that are normally off, but can be turned on (e.g., gfp gene in the pGLO plasmid)

Answer 72

Inducible genes

Question 73

genes that are normally on, but can be turned off

Answer 73

Repressible genes

Question 74

PGLO Plasmid is an example of an

Answer 74

expression vector

Question 75

Recombinant plasmid has origin of replication, multiple cloning site, and selectable marker (antibiotic resistance gene), AND promoter sequence for the foreign gene Inside the cell, plasmid is replicated and the foreign gene is expressed (Protein of the foreign gene is produced)

Answer 75

Expression vector

Question 76

Recombinant plasmid has origin of replication, multiple cloning site, and selectable marker (antibiotic resistance gene), but no promoter sequence for the foreign gene Inside the cell, plasmid is replicated but the foreign gene is not expressed

Answer 76

Cloning vector

Question 77

How to make cells competent fortransformation → In nature → In the lab

Answer 77

→ In nature, competent cells are those that can take up plasmid/DNA from the environment → In the lab, we are artificially making cells competent so that they can take up the recombinant plasmid

Question 78

what does the Ca2+ in CaCl transformation solution does?

Answer 78

Ca2+ shields charges on DNA to make it less polar

Question 79

is like chicken noodle soup for bacteria. It has all the nutrients bacteria need to grow:

Answer 79

LB (Lysogeny broth or Luria Bertani) broth

Question 79

incubation of cells in cold temperature followed by incubation in warm temperature

Answer 79

Heat shock

Question 80

why heat shock to transform cells?

Answer 80

add heat to create pores in the membrane

Question 81

LB (Lysogeny broth or Luria Bertani) broth inclusions

Answer 81

。 Carbohydrates 。 Amino acids * Nucleotides 。 Salts 。 Vitamins

Question 82

why do we CaCl2 transformation solution?

Answer 82

Shields negative charge on DNA → Allows plasmid to more easily bind to and pass through the cell membrane

Question 82

Transformed cells (with the plasmid) will make _______ , which breaks down ampicillin. This enables them to grow on ampicillin plates

Answer 82

beta- lactamase

Question 82

Transformation summary

Answer 82

1. CaCl2 transformation solution 2. Pre-heat shock incubation on ice 3. Heat shock 4. Post-heat shock incubation on ice 5. Incubation at room temperature with LB broth 6. Spread on culture plates

Question 83

why do we need incubation at room temperature with LB broth

Answer 83

Allows cell recovery and beta-lactamase expression so bacteria can grow on plates with ampicillin

Question 83

why do we need to Pre-heat shock incubation on ice?

Answer 83

Slows fluid plasma membrane for greater shock

Question 84

why do we need to heat shock specimen?

Answer 84

Increases permeability of cell membranes

Question 84

why do we need to post-heat shock incubation on ice?

Answer 84

Restores cell membrane