Bacterial Transformation Flashcards
how can foreign genes be introduced into organisms
- through bacterial transformation
what is the purpose of a plasmid during transformation
a plasmid is a small, circular piece of double-stranded DNA
-plasmid DNA contains coding sequences (genes) which are expressed by the bacterium. Often, the gene carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance
role of the beta-galactosidase (LacZ) (color marker gene)
- breaks down lactose into galactose and glucose
- can also cleave the synthetic analog of lactose, X-GAl
- creates a product that is blue when cleaved with XGal
role of beta lactamase (ampicillin resistant gene)
- it destroys the activity of ampicillin by breaking down the beta lactic ring
- thus, bacteria with beta lactamase gene can resist ampicillin
purpose of nutrient agar
to contain the bacteria
purpose of ampicillin
ampicillin is a member of the penicillin family in antibiotics
- destroys the activity of bacteria by destroying its cell wall
- ampicillin contains a chemical group called BETA LACTAM RING which blocks the process by which bacteria link molecules to create a cell wall
purpose of X-gal
- synthetic analog of lactose that when cleaved by the LacZ gene has a blue product
- allows for the identification of ampicillin-resistant bacteria
purpose of pBlu
-a plasmid with all the necessary criteria to use in the bacterial transformation lab
what is bacterial transformation
when a host organism takes in foreign DNA and expresses its gene
- the introduction of apiece of DNA, like a plasmid, into a bacterial cell
what does a plasmid contain
- origin of replication
- gene for antibiotic resistance (encodes beta lactamase)
- color marker gene (beta galactosidase/lactZ)
- polylinker
what is a recombinant plasmid
a genetically engineered plasmid (with a foreign gene inserted in it)
why do we lower the temperature in the transformation experiment
to slow down the movement of the phospholipid in the membrane of the bacteria to efficiently neutralize the charges
why do we use the heat shock
rapid change in temperature induces the formation of pores in the bacterial membrane so that the plasmid DNA can enter
what are competent cells
when bacteria cells are in a state in which they are able to take up DNA, they are referred to as competent
-transformation rarely occurs naturally, so by subjecting bacteria to certain conditions, we can enable many of them to take up DNA
- what is insulin +relation to bacterial transformation
- diabetes?
- a hormone that originates from the pancreas, regulates glucose in blood
- we can create insulin from bacteria by purifying it
- diabetes consists of type 1 and type 2. It is a disorder that deactivates the insulin
how is a plasmid genetically engineered to contain a gene of choice
-a plasmid has sequences that restriction enzymes correspond to, so restriction enzymes can manipulate the plasmid to create a gene of choice by ‘cutting’ there
why do we use CACl2 (calcium chloride) in the bacterial transformation lab
- Plasmid DNA is a hydrophilllic molecule which will normally pass through the plasma membrane of the bacterium in order to accomplish transformation, the bacteria cells must first become competent to make the plasmid DNA.
- this is done by neutralizing the negative charges of the membrane, the DNA molecule with calcium chloride
origin of replication?
- a sequences of DNA is recognized by DNA replication enzymes
- when a bacterium divides all of the plasmids contained within the cell are replicated, thus each daughter cell receives a copy of each plasmid
Results of bacterial transformation (with plasmid)
- (LB) lawn of bacteria
- (LB +AMP) bacterial growth ( white colonies)
- (LP+AMP+X-GAL) Blue colonies (and white)
Results of bacterial transformation (without plasmid)
- (LB) lawn of bacteria
- (LB +AMP) nothing
- (LP+AMP+X-GAL)nothing
where do transcription and translation take place
transcription: nucleus
translation: cytoplasm
