Difference between revisions of "Team:Heidelberg/Methods test"

Blue Plaque Assay (170801)

1. Introduction

The Blue Plaque Assay is a method to determine M13 phage titer.

2. Material

LB/Amp-Agar Plates (1.5 % agar):
Plates with bottom agar (15 g/L agar in LB medium) supplemented with antibiotic are prepared at least 30 min before plaque assay. Top Agar (0.7 % agar):
Top agar containing 7 g/l agar is autoclaved and stored at ~56 °C in the heating cabinet. 3 ml top agar is sufficient for one agar plate. X-gal stock solution (40 mg/ml):
0.2 g X-gal is dissolved in 5 ml DMSO and stored at -20 °C in the dark. It is useful to aliquot the stock solution in 1.5-ml reaction tubes. Transformed S2060 E. coli strain (ID:47):
S2060 E. coli possess the lacZ gene under control of a PSP promoter. For the Blue Plaque Assay a transformed S2060 strain like E. coli strain ID:47 containing the AP of the S1059 strain (gene III under a PSP promoter) are used. (This protocol can be modified to investigate if the evolving phage can resist stronger selection pressure. Therefore S2060 strain has to be transformed with the associated AP and the antibiotics has to be adapted)

3. Procedure

Day 1: S2060 AP_PSP_gIII cells (ID:47) or the appropriate cell type are used to inoculate 4 mL of 2xYT medium containing 25 mM glucose and carbenicillin or the necessary antibiotic for that cell type.
The culture is placed in the 37°C shaker in the morning and are grown until an OD600 of 0.6-0.8. In the meantime, 10 to 10²-fold serial dilutions of the phage suspension are prepared with LB medium. 1 ml final volumes are convenient. To prevent cross-contamination filter tips must be used. The phage samples can be stored at 4 °C for a long time and still be able to infect E. coli cells. Shortly before the E. coli culture reaches the appropriate OD600 top agar (3 ml per plate) is transferred to a 50-ml falcon and placed in a preheated water bath at 50 °C. Top agar is then supplemented with the necessary antibiotic and the x-gal stock solution (40 mg/ml). For 10 ml top agar:

• 10 µl antibiotic (1000x)
• 100 µl X-gal stock solution (100x)

As soon as the E. coli culture reach an OD600 of 0.6-0.8, cell suspension is gently mixed by resuspension with a pipette and 50 µl are transferred to a 1.5-ml reaction tube for each condition. The following steps has to be performed quickly and separate for each condition. Phage suspension is shortly vortexed and 1 µl is pipetted to the E. coli culture. 700 µl of the top agar is thorough mixed with the cell and phage suspension. This is immediately poured on the corresponding quarter of the plate and rocked gently to evenly spread it. After allowing the top agar to solidify for few minutes, plates were sealed with parafilm and incubated overnight at 37°C. Day 2: On the next day blue plaques can be counted. For quantification of the phage titer the number of plaques should be between 10 and 100.
The amount of phages in the solution is calculated by: PFU/ml = number of plaques x dilution factor x 1000 e.g. 12 blue plaques are counted on a plate quarter where 1 µl of a 10^-4 phage dilution was plated.
12 x 10⁴ x 1000 = 1.2 x 10⁸ PFU/ml

Mutagenesis Plasmid Activity Assay

To figure out to which extend the mutagenesis levels of the Mutagenesis Plasmid (MP)
1, MP4 and MP6 differ from each other and are functioning as expected from literature, an mutagenesis activity assay is performed. MP1, MP4 and MP6 All MPs contain an arabinose inducible promoter P_BAD. Upstream of P_BAD the araC protein is
encoded in the opposite direction, it regulates the activity of the P_BAD promoter. The
expression cassette downstream P_BAD consists of multiple mutagenesis supporting elements.
MP6 shows the strongest mutagenesis level (Development of potent in vivo mutagenesis plasmids with broad mutational spectra, Badran et al., 2015) followed
by MP4 and MP1. Development of Antibiotic resistances Due to the exceptional high mutagenesis level, E. coli cells carrying one of the previously
described MPs, quickly develop resistances. To measure the mutagenesis level and the linked
antibiotic resistance development, E. coli cells with MP1, MP4 and MP6 as well as a negative control
are plated on agar plates with different concentrations and types of antibiotics.
After 18-21 hours of incubation at 37 °C the numbers of colonies are counted. To carry out this experiment, please follow the detailed instructions below: Note, that all experiments are performed in triplicates

1. Preparation of the agar stabs
   • plate cells from each agar stab on an agar plate containing 100 mM Glucose (to inhibit the MPs)
   • After 8-12 hours of incubation at 37 °C pick a single colony and follow further steps below
2. Inoculate an overnight culture of 4 ml 2xYT medium with 200 mM arabinose (to induce the MPs) and
   1000-fold dilution of 34 mg/ml chloramphenicol stock (resistance of all MPs)
3. Incubate the overnight cultures at 37 °C for 18-21 hours
4. Prepare 2xYT agar plates with 100 mM glucose + antibiotic. Label all plates properly: name of antibiotic +
   dilution
5. Plate 50 µl (do not centrifuge) directly on the appropriate plate for undiluted samples and
   dilute 10-fold or 100-fold diluted samples with 2xYT medium and then plate the diluted samples.

Each plate is divided into three sections.

• undiluted
• 10-fold diluted
• 100-fold diluted

1. Incubate agar plates at 37 °C for 18-21 hours
2. Count the number of colonies on each plate and fill in your results in the
   following table:

Please take photographs of all plates and make sure that the labelling is visible.