Bacterial Transformation

What is a plasmid?

a small circular piece of DNA found in bacteria that can be used in transformation to introduce new genes (such as antibiotic resistance)

What are the parts of a plasmid?

origin of replication: allows initiation of replication

antibiotic resistance gene: key for transformation because it can also serve as a marker to see if the bacteria took up plasmid

promoter: drives transcription of the targeted gene

What is the difference between a bacteria's plasmid and the one chromosome?

chromosome: essential to bacteria, circular and supercoiled for space in the cell

plasmid: NOT essential, small round DNA molecule that replicate independently

What is a recombinant plasmid?

a plasmid containing a cut out region replaced with another with a specific desired gene (plasmid made by combining two different sources of DNA)

How are restriction enzymes used to create a recombinant plasmid?

restriction enzymes cut genes from one organism out then cut out the area to insert in from the plasmid with the same restriction enzymes; they cut both unevenly which result in "sticky ends" due to the complementary base pairs that are then stuck together to insert the genes into the plasmid

What are the steps of bacterial transformation?

1. desired gene from a cell is cut out with restriction enzymes

2. the plasmid is also cut open with the restriction enzymes unevenly, resulting in the sticky ends

3. the desired gene binds to the plasmid with the corresponding sticky ends

4. the transformed plasmid is inserted into a new bacteria cell, becomes part of the DNA

5. a genetic marker tells if the bacteria cell has successfully taken up the foreign DNA

What is a gene of interest? Its role?

the specific gene targeted to be inserted into a plasmid; when inserted, results in recombinant DNA that can then be inserted into bacteria to have that trait

What are sticky ends?

the uneven ends that result from the staggered restriction enzyme cuts that can then be stuck together with complementary base pairing to bind a gene to a plasmid

What is a genetic marker? Examples?

a gene in a plasmid that tells if the bacteria has successfully taken up the plasmid and desired gene by testing it; can include antibiotic resistance because if it survives/grows in antibiotics, it took up the gene of interest as well or dyes (visible)

What is an activator protein?

a protein that regulates transcription by binding near the promoter and thus enabling RNA polymerase to initiate transcription of the desired gene (ex: araC)

LB

food for bacteria that promotes its growth

Ampicillin

antibiotic used in the lab to confirm successful plasmid transformation by the bacteria's growth, kills bacteria by breaking the formation of cell walls

Arabinose

sugar that allows araC to enable transcription for rfp, thus allowing for proteins that result in the red/pink color trait

Red fluorescent protein

protein coded by the rfp gene, used in lab to confirm the transformation and transcription of the E. coli because it makes it red or pink; is only effective in the presence of arabinose

What are competent cells and how are they prepared?

cells that are prepared to take up foreign DNA efficiently by being treated with calcium (to neutralize the cell membrane's negative charge to let DNA through) and heat shocked (to create a pressure gradient outside to allow plasmids to enter)

What is the difference between colonies and lawns of bacteria?

lawns: continuous cover of bacteria on the surface over entire medium, abundance of bacteria growth

colonies: dots scattered over medium; millions of bacteria in each dot

What is the plasmid used to transform E. coli? (in lab) What are the parts of it?

pARA-R plasmid; araC, promoter pBAD, rfp, ampR

What enzyme does arabinose help activate to transcribe the rfp gene?

RNA polymerase

Why can the sticky ends be stuck together?

because the same restriction enzymes are used to cut the desired gene and the plasmid

What is the purpose of growing bacteria in ampicillin when it has been transformed?

if the bacteria has successfully taken up the plasmid with the ampR, then it will survive. this tells that the bacteria has taken up the plasmid, and thus that it has the rfp, or gene of interest (ampR is the genetic marker)

What is transformation?

the process of a cell taking up foreign (recombinant) DNA to change its original DNA to have a specific desired trait

What is a transgenic organism?

an organism that contains genes from other species

Why is it important to have multiple copies of a recombinant plasmid in a cell for transformation?

in order to increase the chances of the cells successfully taking up the plasmid and thus carrying the desired gene, as only very few cells can take up the foreign DNA. having more=more chances and higher protein production for the desired trait through protein synthesis and replication

What factors help the rfp be expressed as a trait in the last plate?

ampC turns on the promoter for rfp in the presence of arabinose, as it is an activator protein. this allows RNA polymerase to bind to the promoter and start transcription of the rfp gene and is then translated into proteins that cause the red/pink color (trait)

What are the similarities and differences in DNA among different organisms?

similarities: dna replication, stores genetic info, structure, bases, double helix

differences: location, number of chromosomes, sequence of bases, introns, genes