Introduction

General Information

Motivation

Procedure

As it is important to prevent phage contaminations at any time, all the following PREDCEL steps have to be performed with filter tips. Benches should be cleaned with 10% H202 (pay attention with handling) or another suitable solution for phage inactivation. If practicable, use UV light on benches and in incubators to sterilize after and before your experiments.

Step 1: F+ strains
After successfully transforming your AP and MP plasmids into your bacterial strain (see transformation protocols), make sure the used strain carries the F-Pilus plasmid as it is needed for proper M13 phage infection. Therefore, grow a bacterial culture from a picked single colony. This culture should be plated on a tetracycline (tet) plate, since the strain should carry a tetracycline resistance on its F-Pilus plasmid.

Step 2: Prove MP to work
Single colonies showing a positive result in should be picked and grown in 2YT medium, supplemented with the appropriate antibiotics and 100 mM glucose. Glucose must be added as it prevents the induction of the pBad promotor on the MP and thereby the mutagenesis. The resulting bacterial growth should grow until an OD600 of 0.6 - 1.0 is reached. Following this, the functionality of the MP should be tested before starting your PREDCEL run. Therefore, follow instructions of the protocol. The evaluation of your AP should take place before starting your PREDCEL run as well (see ).

Step 3: Glycerol stocks
Based on a positive result in , glycerol stocks from the prepared bacterial culture should be prepared. In order to do that, a main stock of 2 ml and several aliquots of 100 µl in PCR tubes should be prepared (see respective protocol) and stored at -80°C.

Step 4: Contamination test
Before finally starting the PREDCEL run, the bacterial culture and thereby the glycerol stock should be tested for a phage contamination. Therefore, plaque assays with the supernatant of the culture should be implemented following the . If the results are showing no plaques, your PREDCEL run can start.

Step 5: Centrifugation and MP activation
Starting your PREDCEL run, a new culture should be grown, using one of the 100 µl glycerol stock aliquots for inoculation. This culture should also reach an OD600 = 0.6-1.0 and has to be prepared early enough to minimize time between gain of phage supernatant and new infection round. If the culture reaches the respective OD600 too fast, dilute until you can infect with phages or cool on ice if it takes less than 10 minutes until infection. Centrifuge 10 ml or more of your culture for 10 min at 3750 g at room temperature to pellet your bacteria. Afterwards, resolve your cell pellet in a 150 ml flask with a volume of 2YT equal to the volume spun down containing 100 mM arabinose to induce MP.

Optional: Keep 1 ml of culture supernatant after centrifugation and 1ml of medium solution you dissolve with. Store both at 4°C. Additional to PCR they could later be used for contamination check by plaque assay.

Step 6: Phage infection
In the next step, infect the resuspension with a suitable number of phages. As titers of inoculation phage can vary a lot a multiplicity of infection (MOI) of 1 is recommended for PREDCEL, rather than giving a specific inoculation volume. MOI of 1 means that there is one phage per cell in solution. In contrast, while performing PREDCEL, define a certain phage supernatant volume you kept at 4°C to be transferred, for instance 1 ml, as phage titers cannot be determined fast enough by plaque assays to calculate the right MOI. Make sure you have at least 1 ml of phage supernatant left to analyze. Transfer the same number of phages used for infection into a phage buffer solution, especially if titer of inoculation phage is unknown.

Grow the infected cultures for 1 to 24 hours, respective to your propagation results during the AP testing, at optimal growth conditions (for choose 37°C and about 220 rpm).

Step 7: Phage supernatant
After incubation time spin down at least 2 ml of cells at 6000 g for 3 min at room temperature to pellet your cells. Phages in the supernatant are now separated from the bacteria in the pellet. Store phage supernatant at 4°C. Discard pellets and the rest of the culture.

From now on repeat steps 5 to 7. After one round is completed you can pause the PREDCEL process and restart later with the stored supernatant.

While PREDCELing, test PCRs with specific products for your SP should be performed as well as test plaque assays should be implemented to detect and prevent phage washout or phage contamination in your used stocks, media and bacterial cell cultures.
If contamination or wash out is recognized, take the last sample proven to contain phages and which is free of contamination to start a new PREDCEL iteration round with a fresh culture.

Step 8: Plaque assay
Perform plaque assays of your PREDCEL samples including inoculation phage and culture samples from (see protocol) to calculate phage titers and check for washout.

Step 9: Sequencing
To check your evolutionary progress, at last eight plaques should be picked to use them for insert amplification via PCR. Afterwards, sequencing of your PCR product can be implemented with insert specific sequencing primers. Mention that mutations taking place while PREDCEL may lead to inefficient primer annealing.

Troubleshooting:
As phage propagation rate and mutation efficiency usually varies between different gene circuits and phages, but also due to the achieved changes in activity within one PREDCEL run, you might have to adopt several parameters to achieve a lower selection pressure (if phages get lost over time), a higher selection pressure (only random mutations occur) or changes in mutation rate (too little/many mutations). These parameters are:

• the amount of culture volume to be infected and the corresponding flask size
• the time of incubation after infection
• the amount of phage supernatant transferred

To check for the right parameters pre-experiments, need to be performed for an optimized propagation efficiency.

You should also prepare several strains of your bacteria carrying APs with different RBS-strengths and origins of replication and different MPs (MP1/4/6 vary in mutation rate).

If you have problems with phage propagation due to no or too little starting activity an initial drift phase without selection pressure might help in the beginning.

Another helpful adaption is the usage of a helper-culture. This culture should be used between every or several PREDCEL AP-iteration rounds and must provide geneIII expression that is only coupled to phage infection. Thereby all phages that are transferred can reproduce to gain a high phage titer that might be needed to prevent phage washout. As previously phages showing higher activity propagated faster than those showing no or low activity they will, as a consequence, achieve higher phage titers while this helper-culture-phase. Thus, helper-culture phase should not redeem previously achieved selection for beneficial mutations.

Note: Contamination in theory must not a priori lead to end of current evolutionary process as contamination phages should not be able to propagate on your AP-carrying host cells if it lacks geneIII. Nevertheless, don't go on with PREDCEL when contaminated in order to gather valid data, especially regarding your phage titers. This counts even more if your AP-design might allow other non-wild-type phages to propagate.