Inducible Plaque Forming Assay

1. Introduction

Our previous assay revealed that the M13 phage genes and the M13 origin worked together with the pSB origin. Next, we confirmed whether the release of the M13 phage particle depends on the induction of g2p expression. We inserted lux promoter (which is activated by 3OC6HSL-LuxR complex) upstream of g2p, as an inducible promoter (Fig. 3-4-1)（See more about our plasmid construction). The method for our assay is briefly shown in Fig. 3-4-2. First, we cultured E. coli to which was introduced our new part ([http://parts.igem.org/Part:BBa_K1139021 BBa_K1139021]), and added 3OC6HSL, which induces the phage release. Second, we centrifuged the culture and obtained phage particles. Finally, we added the phage particles and lawn culture (E. coli containing pSB6A1-Ptet-luxR) to the soft agar, and poured it on a YT plate. Moreover, we confirmed that the inducible release of M13 phage was also realized with BAD promoter.

Fig. 3-4-1. Our plasmid construction

Fig. 3-4-2. How to perform our inducible phage release assay

2. Materials and Methods

2-1. Plasmid construction

pSB3K3-Plux-M13-Plac-GFP ([http://parts.igem.org/Part:BBa_K1139021 BBa_K1139021])

pSB6A1-Ptet-luxR ([http://parts.igem.org/Part:BBa_S03119 BBa_S03119])

pSB3K3-Pbad-M13-Plac-GFP ([http://parts.igem.org/Part:BBa_K1139026 BBa_K1139026])

pSB6A1-Ptet-araC ([http://parts.igem.org/Part:BBa_I13458 BBa_I13458])

2-2. Strains

DH5alpha (E. coli of high competence)

JM109 (F+ strain E. coli)

2-3. Media

LB

Bacto tryptone	10 g/L
Yeast Extract	5 g/L
NaCl	10 g/L

YT soft agar

Bacto tryptone	8 g/L
Yeast Extract	5 g/L
NaCl	5 g/L
Agarose	6 g/L

YT plate

Bacto tryptone	8 g/L
Yeast Extract	5 g/L
NaCl	5 g/L
Agarose	15 g/L

2-4. Others

3OC6HSL dissolved in DMSO (>100 microM)

Autoclaved pieces of filter paper (about 1.5 cm in diameter)

2-5. Protocol

Preparation
1. Transform DH5alpha with pSB3K3-Plux-M13-Plac-GFP and pSB6A1-Ptet-luxR.
2. Prepare overnight cultures of the transformed DH5alpha and JM109, DH5alpha in LB containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL).
3. Take 30 microL of the overnight cultures of the transformed DH5alpha into 3 mL LB medium containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) . (->fresh cultures)
4. Incubate the fresh cultures at 37°C for 2 h.
5. Add 3 microL of 5 microM 3OC6HSL in DMSO (final concentration of 5 nM) to the fresh cultures and incubate them at 37°C for 4 h.
6. Centrifuge the overnight cultures of the transformed DH5alpha at 9,000g for 1 min.
7. Pipette the supernatant into a 1.5 mL tube.
8. Dilute it 100 times with water. (-> phage-particle-solution)
   Plaque formation
9. Transform JM109 with pSB6A1-Ptet-luxR
10. Prepare overnight cultures of the transformed JM109 at 37°C.
11. Melt the YT soft agar by using a microwave.
12. Add ampicillin to the YT soft agar.
13. Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
14. Dispense 400 microL of the overnight cultures of the transformed JM109 to a 1.5 mL tube.
15. Into the 1.5 mL tube, add 100 microL of the phage-particle-solution.
16. Transfer all the content of the 1.5 mL tube to the dispensed soft agar, mix them, and spread all of it on a YT plate.
17. Wait for the YT soft agar to solidify at room temperature (for about 5 min.).
18. Put an autoclaved piece of filter paper on the plate, and drip 20 mL of 3OC6HSL in DMSO (100 microM, 5 microM or DMSO only) into the piece of filter paper.

3. Results

The result of the plaque forming assay is shown in Fig 3-4-3, 3-4-4. On the plate, the plaques were formed at some distance from the piece of filter paper. The result shows that the phage release is regulated by the induction of 3OC6HSL. We also analyzed the distribution of the plaques (Fig. 3-4-5, 3-4-6). The histogram result implicates that the plaques are formed only at the appropriate concentration of 3OC6HSL in the medium (Fig. 3-4-7). In the neighborhood of the piece of filter paper, the expression of g2p is so activated that the phage particles cannot be produced efficiently, because the production of the single stranded DNA largely exceeds that of the coat proteins. In contrast, far from the piece of filter paper, the expression of g2p is so activated that the phage particles cannot be produced efficiently, because the production of the single stranded DNA isn't enough to produce phage particles.

Fig. 3-4-3.

Fig. 3-4-4.

The plaques were formed using the JM109 (with pSB6A1-Ptet-luxR) overnight cultures and the phage-particle-solution. The phage particles were obtained from the supernatant of the fresh cultures (+3OC6HSL) of transformed DH5alpha (with pSB3K3-Plux-M13). A mixture of 3.5 mL YT soft agar, 100 microL of 0.01X supernatant and 400 microL of the overnight cultures of JM109 (with pSB6A1-Ptet-luxR) was poured on a YT plate. After solidifying the soft agar, we put a piece of filter paper on the plate and dripped 3OC6HSL in DMSO (or DMSO only) on the piece of filter paper.

Fig. 3-4-5.

Fig. 3-4-6.

Fig. 3-4-7. The distribution histogram of the plaques(Fig. 3-4-6)
This histogram shows distance from the piece of paper in a horizontal axis, and shows number of plaques over distance squared, which means the rate of plaque formation, in a vertical axis. The bin range is 0.2 cm.

We hypothesize that in the vicinity of the piece of filter paper, the expression of g2p was too activated to produce the phage particles efficiently, because the production of the single stranded DNA largely exceeded that of the coat proteins. In contrast, too far from the piece of filter paper, the expression of g2p was not so activated that the phage particles could not be produced efficiently, because the production of the single stranded DNA wasn’t enough to produce phage particles. The plaques were formed only when the inducer existed in the medium at appropriate concentrations.

To demonstrate the hypothesis is true, we changed the waiting time before dropping AHL from 5 min. to 2h. In the meantime, phage coat proteins would be sufficiently produced in the lawn cells. Fig. 3-4-8 shows that the plaques were formed on the whole surface of the plate. From this assay, we can conclude that the balance of the production of coat proteins and single stranded DNA is very concerned with phage release.

Fig. 3-4-8. Formed plaques after we changed the waiting time prior to dropping AHL

We also confirmed that inducible phage release would be realized with another promoter. We used BAD promoter(arabinose-inducible promorter) for g2p. The result of the arabinose-inducible plaque forming assay is shown in Fig 3-4-9, 3-4-10, 3-4-11. The result shows that the phage release is regulated by the induction of arabinose. We confirmed that we can use two kinds of promoters with our part (BBa_K1139022). By replacement of the promoter upstream of g2p with various promoters, we have constructed a part collection of M13 phage. We have not only constructed the part collection, but also assayed the function of parts.

Fig. 3-4-9. Formed plaques with arabinose and araC⁺ receiver

Fig. 3-4-10. Formed plaques without arabinose and with araC⁺ receiver

Fig. 3-4-11. No plaques with arabinose and without araC⁺ receiver

Plaques were formed with arabinose and araC⁺ receiver (Fig. 3-4-9). Even though we did not add arabinose, plaques were formed araC⁺ receiver (Fig. 3-4-10). However, no plaques were formed with arabinose and without araC⁺ receiver (Fig. 3-4-11). The reason we thought why plaques were formed with no arabinose and araC⁺ receiver is that too much araC expressed in receiver led to leaky expression of g2p that is enough to form plaques (Fig. 3-4-12).

Fig. 3-4-12. Summary of the result of the arabinose-inducible plaque forming assay