CM13 Interference-Resistant Helper Phage

CM13 - Interference-Resistant Helper Phage

Technical Description

CM13 is a helper phage especially engineered for phage display. CM13 is a derivative of M13KO7 (1) containing the interference-resistant ir3B A->G mutation (2) at position 8418 of M13KO7. CM13 is equivalent to VCSM13 helper phage and can be used as a direct replacement. CM13-infected cells often produce more virions than M13KO7 while giving similar levels of display on the minor coat protein III (learn about CM13 here). This helper phage makes preparation of virions particularly efficient at small scale, thus facilitating the overall screening process. This preparation contains enough helper phage to superinfect up to 1 L (1000 ml) of bacterial culture.

Figure 1. Production of phagemid virions by CM13. pADL-23/HyHEL-10 scFv containing TG1 cells were grown to 1 O.D. (turbidity taken at 600 nm) and transduced with varied amounts of helper phage; after o/n incubation, phagemid virions were prepared by PEG precipitation and quantified by U.V. spectrophotometry; results are expressed in virions per ml of bacterial culture. In this experiment, maximum transduction is reached for CM13 at 1 x 10(9) pfu/ml while larger amounts are needed for M13KO7; CM13 always produced more phagemid virions.

Applications

• Phage display.

For research use only; not intended for any animal or human therapeutic or diagnostic use.

Source

CM13 virions were isolated from the supernatant of infected E. coli TG1 cells and purified by PEG precipitation.

Specifications

Composition: 50% glycerol TBS buffered.
Concentration: 2 x10¹² pfu/ml; 1 x10¹³ virions/ml; infectivity ~20%
Storage temperature: -20°C.
Product size: 1 ml.

Quality Control & Certification of Analysis

Virion concentration:

Phage DNA concentrations are determined by UV spectrophotometry and virion concentrations calculated based on the length of CM13 genome.

Phage titer:

CM13 preparations are serially diluted and mixed with melted LB top agar and TG1 cells. The mixtures are poured over LB bottom agar plates and incubated overnight at 37°C; the morning after plaques are counted. Controls include plates without virions and have no visible plaques.

Infectivity:

Infectivities are counted as the ratio between phage titer and virion concentration. Infectivities between 15 and 20% are expected.

Gene II ORF sequence:

ORF DNA is amplified by PCR and sequenced.

Absence of defective interfering (DI) particles:

Integrity of phage particle ssDNA is verified by electrophoresis.

Certification:

Products meet all specifications.

Notes

Conditions for optimal transduction:

Always use bacteria freshly made from the night before; typically grow your phagemid-containing bacteria overnight in 2xYT medium from a single colony in the presence of ampicillin and glucose at 30°C or 37°C. Dilute the bacterial culture 1:20 v/v with fresh 2xYT medium and incubate for one hour at 37°C. Measure the absorbance at 600 nm, at best using a large 1 ml cuvette and a 1:5 or a 1:10 dilution; adjust to 0.5 OD and add CM13 between 1 x 10(9) and 1 x 10(10) pfu/ml. We recommend 2 x 10(9) pfu/ml or 1 µl of CM13 preparation per ml of culture as a good balance between not using too much helper and achieving a high level of superinfection; more helper may yield more phage but results are often inconsistent.

Immediately transfer the culture to a shaker and incubate for 30 min to 1 h at 37°C and 250 rpm; preincubation of helper and bacteria on the bench or at 37°C without shaking is unnecessary and often leads to a greater variability in phage yields; best superinfections are obtained at 37°C and 250 rpm. Then add kanamycin 50 µM, ampicillin 100 µM and IPTG 200 µM if you are using pADL-10b, pAK100 or pAK200 phagemid and lower the temperature to 30°C; harvest the phage after 8 h to o/n.

Influence of cell density:

An F⁺ bacterial strain containing the F plasmid is required for superinfection by CM13 virions. Removal of glucose, induction of the Lac promoter and expressing the fusion coat protein at possible toxic levels may cause the bacteria to stop growing; therefore, it is better to add the helper phage at the highest cell density possible. Inversely the presence of a large percentage of non-transduced bacteria can lead to low phage production; this problem is accentuated by a natural immunity to M13 superinfection caused by endogenous expression of full-length protein III, including expression by the phagemid. We have determined that the optimal cell density at which the helper phage is added to maximize phage production is at an optical density between 0.4 and 0.5 at 600 nm for TG1 and SS320 cells. In these conditions, use of a truncated protein III to limit immunity to superinfection is not necessary.

References

1. Vieira, J. and Messing, J. (1987) R. Wu and L. Grossman (Eds.), Methods Enzymol., 153, pp. 3-11. San Diego: Academic Press.
2. Enea, V. and Zinder, N. (1982) Interference resistant mutants of phage f1, Virology 122, pp. 222-226.

Related Products

M13KO7 Helper Phage