Transformation efficiency

The double deletion of the constitutive alanine racemase (alr) and the catabolic alanine racemase (dadX) in the DH5α and KRX strain, resulting in the phenotypes KRX Δalr ΔdadX and DH5α Δalr ΔdadX respectively. This leads to a strict D-alanine auxotrophy, which can be complemented by a plasmid carrying one of the alanine racemases or D-alanine supplementation in the media.
The plasmid BBa_K1465401 which is used for the complementation carries the constitutive alanine racemase alr and the chloramphenicol acetyltransferase for the chloramphenicol-resistance (Cm). Besides the plasmid contains RFP BBa_J04450 as a reporter for the characterization of the complementation efficiency.
To investigate the antibiotic-free selection the plasmid was transformed in different concentrations into electrocompetent cells of DH5α Δalr ΔdadX. Due to the fact, that the cell can store D-alanine for a certain periode of time the incubation of the cells after the transformation was performed in normal SOC-media without supplementation and in SOC-media with supplementation of 3 mM D-alanine to maintain the cell viability of the transformants before they are able to express the alanine racemase which is essential for the accumulation of D-alanine and bacterial growth. After the incubation the cells were stread out either onto LB plates for the antibiotic-free selection via complementation of the D-alanine auxotrophy or onto LB plates containing 30 mg/L chlormaphenicol as control. The successful transformation can be verified by the red color of the reporter RFP, while false-positive colonies remain white.
An example for the transformation of 0.33 ng plasmid BBa_K1465401 into the E. coli strain DH5α Δalr ΔdadX is shown in Figure 3. The upper row shows different volumes from 50 µl, 100 µl and 200 µl were spread onto LB containing chloramphenicol and the lower row shows the same volumes were spread out onto normal LB for the selection without any antibiotic. It appears that the antibiotic-free selection is more efficient than the selection with chloramphenicol because more colonies were observed on each plate with equal volume plated.

Figure 3: Colony forming units (cfu) for the selection with chloramphenicol (upper row) and antibiotic-free selection (lower row). 0.33 ng of the plasmid BBa_K1465401 was transformed into the E. coli strain DH5α Δalr ΔdadX, incubated in SOC-Media supplemented with 3 mM D-alanine and spread out in different volumes from 50 µl (left), 100 µl (middle) and 200 µl (right).

Figure 3 and 4 show the colony forming units (cfu) for the transformation of 0.52 ng plasmid BBa_K1465401 at one hour of incubation in either normal SOC-medium or SOC-medium supplemented with 3 mM D-alanine. First of all it is possible to see, that the incubation of the transformation can be performed without the supplementation of D-alanine (blue bars), even though there are not as many colonies compared to the incubation with 3 mM D-alanine (red bars). Besides, the graphics show that in the absent of D-alanine more false-positive phenotypic white colonies occur (cyan bars) compared to the samples with supplementation of D-alanine (purple bar). In some cases the absence of D-alanin also leads to huge contamination an higher amount of false-positive colonies than correct colonies. This effect can be clearly seen by spreading 200 µl of the transformants incubated in SOC-Media without D-alanine.
The most interesting aspect turns out by the comparison between the chloramphenicol selection and the antibiotic-free selection (AB-free). It can be clearly seen that the amount of positive red colonies is 2.88 ± 0.45 (3 mM D-alanine supplementation) and 5.91 ± 1.53 (without D-alanine) fold higher compared to the selection with the antibiotic chloramphenicol. This might be due to the active inhibition of chlormaphenicol, whereas the lack of D-alanine can be compensated by intracellular storage of D-alanine or eventually by temporary grow arrest, until the alanine racemase is expressed.

Figure 4: Colony forming units (cfu) for the selection with Chloramphenicol. 0.52 ng of the plasmid BBa_K1465401 were transformed into the E. coli strain DH5α Δalr ΔdadX and incubated in normal SOC-Media or SOC-Media supplemented with 3 mM D-alanine. After one hour different volumes were spread onto LB plates containing 30 mg/L chloramphenicol (n = 2 x 2).

Figure 5: Colony forming units (cfu) for the antibiotic-free selection with LB. 0.52 ng of the plasmid BBa_K1465401 was transformed into the E. coli strain DH5α Δalr ΔdadX and incubated in normal SOC-media or SOC-Media supplemented with 3 mM D-alanine. After one hour different volumes were spread onto normal LB plates (n = 2 x 2).

Another important aspect of an antibiotic-free selection approach is the quotient of false-positive and particularly the portion of revertants which could also reduce this quotient in an antibiotic-free selection. Therefore, the ratio of false-positive transformants (white color) to the positive transformants (red color) are shown in Figure 6. Besides, it should be mentioned that the false-positive transformants in the antibiotic-free selection can be also observed for chloramphenicol selection. These false-positive transformants could be bacteria with a spontaneous chloramphenicol resistance, contamination by bacteria with a chloramphenicol resistance or bacteria carrying a mutation within the expression of RFP (BBa_J04450) leading also to a white phenotype. Within the antibiotic-free selection an average portion of 2.83 % ± 0.09 false-positive transformants has been identified, while the antibiotic-selection with chloramphenicol only shows an average of 1.47 % ± 0.77 false-positive transformants. The slightly higher rate of false-positive transformants of the antibiotic-free selection might due to some revertants which are able to accumulate D-alanine without any alanine racemase by mutation and side reaction of another enzyme. Because of the higher transformation efficiency this effect is actually negligible.

Figure 6: Portion of false-positive colonies of antibiotic selection using chlormaphenicol (red bars) and the antibiotic-free approach (orange bars). The false positve colonies have been identified by their white phenotype. For the antibiotic selection an average of 1.47 % ± 0.77 false-positive colonies was investigated, whereas the portion amount for the antibiotic-free selection system is of to 2.83 % ± 0.09.

To obtain more reliable data, the plasmid BBa_K1465401 was transformed in different concentrations (0.33 ng; 0.52 ng and 0.65 ng) and transformed into the D-alanine auxotrophic E. coli strain DH5α Δalr ΔdadX in various volumes (50 µl; 100 µl and 200 µl) to estimate a valid transformation efficiency by using equation (1):
Transformation efficiency [cfu/ng] = colony forming units / (Spread volume [µl]/total volume [µl])*Amount of Plasmid-DNA [ng]
The estimated transformation efficiency for the transformation of 0.52 ng of BBa_K1465401 for the distinct volumes is shown in Figure 7 and the average for the transformation of different amounts of DNA is shown in Figure 8. The result emphasizes, that the antibiotic-free selection is significantly more efficient than the selection with the antibiotic chloramphenicol due to the inhibition of E. coli by the antibiotic. Another important aspect is the reduced transformation efficiency without the supplementation of D-alanine due to the lytic effect when D-alanine is lacking and the cross-linkage of the peptidoglycane can not be performed by D-alanin lacking cells. Even though some cells show a growth.

Figure 7: Comparision of the transformation efficiency for the transformation of 0.52 ng BBa_K1465401. The incubation was performed either in normal SOC media or in SOC media supplemented with 3 mM D-alanine and the colony forming units were counted on LB_Cm as well as normal LB for the antibiotic-free selection (n = 2 x 2).

Figure 8: Comparision of the transformation efficiency for different amount of the plasmid BBa_K1465401. The incubation was performed either in normal SOC media or in SOC media supplemented with 3 mM D-alanine and the colony forming units were counted on LB_Cm as well as normal LB for the antibiotic-free selection (n = 2 x 2).