Bacterial transformation + producing insulin

Question 1

how does Bacterial transformation occur

Answer 1

Genes can be inserted into bacteria, via recombinant plasmids in order for the gene to be expressed and the resultant protein

• the process of adding a recombinant plasmid into a bacterial is called transformation

Question 2

Techniques for transferring plasmids into bacterial cells

Answer 2

once the plasmid has been made, it has to be transferred into cells although the success rate is generally low
- Electroporation
- Heat shock

Question 3

Electroporation

Answer 3

cells are placed into an electric field, given a brief shock that appears to create holes in other membranes to allow plasmid entry

Question 4

Heat shock

Answer 4

heat shock the bacterial cells:
- they are firstly suspended in an ice-cold salt solution
-Then put in 42 degrees solution for 1 MINUTE
- this appears to increase the fluidity of the plasma membrane

Question 5

Antibiotic resistance

Answer 5

Plasmids contain an antibiotic resistance (antibiotic = kills bacteria) gene.

• this will give transformed bacteria a special property (such as antibiotic resistance) above untransformed bacteria.
• when growing on an agar plate that has an antibiotic gene, only the bacteria that have taken up the plasmid will survive other bacteria are still vulnerable to the antibiotic

Question 6

Process of transformation

Answer 6

1) Bacteria and recombinat plasmid are mixed together

2) Some bacteria will be successfully transformed, most will not be

3) Bacteria are grown on the agar plate containing specific antibiotics - the bacteria that have been selected is because of their resistance to the antibiotic

Question 7

negative amp/ - pGLO

Answer 7

expected results: lawn growth, E.Coli should grown unrestrictedly

actual results: there was growth, bacteria is alive

Question 8

negative amp/ + pGLO

Answer 8

expected results: lawn growth
E.Coli +pGLO should grow

actual results: colony growth
some bacteria has survived the process however something went wrong

Question 9

+ amp/ - pGLO

Answer 9

expected results: no growth/ amp killed the bacteria

actual results: no growth

Question 10

+ amp/ +pGLO

Answer 10

expected results: colony growth, those that survived have taken up the plasmid which results in amp resistance

actual results: no growth

Question 11

Producing human insulin

Answer 11

human insulin can be produced using recombinant plasmids and bacterial transformation technology

Question 12

Splicing for insulin

Answer 12

human insulin genes contain introns which are removed prior to translation, However, bacteria do not undergo RNA processing and therefore are unable to remove introns. Insulin genes are inserted into bacteria must first have introns removed

Question 13

Insulin coding

Answer 13

• Mature mRNA coding for insulin can be isolated. The enzyme reverse transcriptase can convert this back into DNA (known as cDNA) without introns.

Question 14

Structure of insulin

Answer 14

human insulin is made up of two polypeptide chains, (quaternary), alpha and beta chains. each chain is coded for a separate gene.

Question 15

process of insulin

Answer 15

• Each insulin gene (A and B) are inserted into plasmids containing antibiotic-resistant genes
• the insulin genes are inserted next to a highly expressed gene (lacZ) which codes for the production of bacterial protein beta-galactosidase
• bacteria are then transformed with the plasmid
• When the insulin proteins are expressed (A and B chains) they are connected to the bacterial beta-galactosidase proteins
• ## the insulin proteins are then separated, the human insulin proteins are purified and then joined together to create the functional insulin protein