Question

Put the following steps in order outlining the transformation of bacteria using plasmid encoding ampicillin resistance and green fluorescent protein:

1. Plate cells on media containing ampicillin.
2. Cell membrane becomes temporarily more permeable to DNA.
3. Treat ampicillin-sensitive cells with electrical shock or exposure to calcium chloride to make them artificially competent.
4. Introduce cells to plasmid encoding ampicillin resistance and fluorescence.
5. Select cells positive for ampicillin.

Answer 1

To carry out the transformation of bacteria using a plasmid with ampicillin resistance and GFP, first make bacteria competent, introduce the plasmid, allow DNA uptake, plate on ampicillin media, and then select for cells with the resistance gene.

Step-by-step explanation:

The ordered steps for the transformation of bacteria using a plasmid encoding ampicillin resistance and green fluorescent protein are:

1. Treat ampicillin-sensitive cells with electrical shock or exposure to calcium chloride to make them artificially competent.
2. Introduce cells to plasmid encoding ampicillin resistance and fluorescence.
3. Cell membrane becomes temporarily more permeable to DNA.
4. Plate cells on media containing ampicillin.
5. Select cells positive for ampicillin resistance.

To explain further:

Treating cells to make them competent means getting them ready to accept foreign DNA. Next, the plasmid is introduced, which carries gene for resistance to the antibiotic and may contain a gene encoding green fluorescent protein as a marker. Making the membrane permeable allows the plasmid to enter the cell. Once transformed, cells are able to grow on media with ampicillin due to the resistance gene. Finally, selective markers such as ampicillin resistance and reporter genes – for instance, one encoding green fluorescent protein – help in identifying the cells that have successfully taken up the recombinant plasmids.