Question

Control Plates

	LB Control Plate	LB + DAP Control Plate	LB + kan Control Plate	LB + DAP + kan Control Plate
Serratia marcescens (recipient)	Pink growth	Pink growth	No growth	No growth
E. Coli/ pRL27 (donor)	No growth	White/creamy growth	No growth	White/creamy growth

Selection/Counter Selection and Efficiency Plates

Media Type	Dilution Factor	Donor/Recipient	Number of Colonies
LB + kan	1	Transformant	129 total; 7 whte/pink colonies
LB + kan	1	Transformant	118 total; 8 white/pink colonies
LB	10^8	Recipient	124 total
LB	10^8	Recipient	148 total

Interpret your results. What was the cell density of succesful transposants? What was the transformation frequency? What was the proportion of transposon insertions that disrupted pigment production?

TF = concentration of transformed cells / total concentration of recipient cells

Pigment Auxotroph Concentration = concentration of pigment auxotroph cells/ concentration of transformed cells

Answer 1

Explanation-

1. In the above experiment, transposon mutagenesis occurs when Serratia marcescens is transformed with pRL27 plasmid. Transposon mutagenesis is a phenomenon where the transposon from the plasmid will be integrated into the host’s genome to induce the mutation. In this question, transposon mutation in Serratia marcescens disrupts the formation of pink colouration in the colonies.

2. It is to be mentioned that the pRL27 plasmid also contain genetic sequences for kanamycin resistance, therefore, Serratia marcescens cells which are transformed will be able to grow in kanamycin plates.

3. On the other hand, the E. coli strain with the pRL27 plasmid require DAP (di-aminopimelic acid) for its growth. This particular auxotrophic E. coli strain requires external source of lysine for its growth. DAP is decarboxylated to lysine, infact, lysine biosynthesis pathway require DAP as an intermediate.

· In condition 1 (control plates), Serratia marcescens is able to grow pink colonies in normal LB (Luria-bertani) agar plates, a common medium used for bacterial growth. However, the E.coli strain shows no growth in normal LB agar plates since it requires DAP for its growth.

· In condition 2 (control plates), Serratia marcescens shows pink colonies in presence of LB+DAP medium. DAP has no effect on Serratia marcescens growth. However, E. coli can grow (forms white colonies) on these plates as DAP is present.

· In condition 3 (control plates), kanamycin (aminoglycoside antibiotic that inhibits protein synthesis in bacteria) is present in the LB agar plates. Serratia marcescens is not kanamycin resistant and fails to grow on these plates. E. coli plasmid pRL27 has kanamycin resistance gene, but due to the unavailability of DAP, E. coli also fails to grow.

In condition 4 (control plates), Serratia marcescens do not grow due to the presence of kanamycin in the plates. This time, E. coli can grow because DAP is available and the plasmid pRL27 contains kanamycin resistance gene.

Calculating cell density of successful transposants-

Successful transposants means the cells or colonies where disruption of pigment has been observed. Two transformants plates have been observed in the experiment. The Serratia marcescens cells growing in LB+kan plates are transformed with plasmid, and code for kanamycin resistance. But, the transposon insertion in S. marcescens genome has occurred inly in few colonies (where the pigment colouration has been disrupted).

The formula of calculating transformation efficiency is given in the question-

Proportion of transposon insertions-