Bacteria transformation Flashcards
1
Q
How can bacteria be transformed into making specific proteins?
A
- Isolate and prepare human genes
- Recombination - inserting GOI into plasmid
- Transformation of bacteria
- Selecting for successfully transformed bacteria with recombinant plasmids
- Extraction and purification of genes
2
Q
What is involved in the isolation and preparation of human genes?
A
- Use the same restriction enzyme as the plasmid (ensure complementary sticky ends increase chance of uptake)
- Ensure introns are removed
3
Q
What is involved in the recombination stage?
A
- Cut plasmid with the same R.E. and mix with GOI and DNA ligase
- Some plasmids will be recombined some wont
- Some proteins will need two groups (group A for a plasmid and GOIA, group B for a plasmid and GOIB)
4
Q
What is recombinant DNA?
A
DNA with multiple sources, doesn’t occur in nature
5
Q
Why do introns have to be removed before the gene is inserted into the bacteria?
A
prokaryotes don’t have introns
6
Q
What is involved in the transformation of bacteria?
A
- Using heat or electric shock
- Opens pores for a small amount of time
- Some bacteria are transformed (take up plasmid) of these some contain recombinant plasmid, some contain non-recombinant plasmid
- Some are not transformed
7
Q
What is involved in the selecting for successfully transformed bacteria with recombinant plasmids stage?
A
- Grow bacteria on an agar plate with ampicillian
- Bacteria without a plasmid die (no ampicillian resistance) therefore selects against the non transformed bacteria
- Grow bacteria in presence of arabinose. This will show blue and white colonies (because of the interuption of the LacZ gene)
- blue = unsuccessful, white = successful
8
Q
What is involved in the extraction and purification stage?
A
- Isolate fusion proteins, cut polypeptide chains off and allow A+B to combine/fold into functional protein
9
Q
What is a vector?
A
10
Q
A
