Real-time observations of single bacteriophage λ DNA ejections in vitro

Grayson et al. 10.1073/pnas.0703274104.

Supporting Information

Files in this Data Supplement:

SI Text
SI Movie General Description
SI Movie 1
SI Movie 2
SI Movie 3
SI Movie 4
SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Table 1

SI Figure 6

Fig. 6. Portions of single DNA ejection events judged as "bad" and not selected for further analysis. From top to bottom: a genome that is stuck to the coverslip at both ends and folded in half, with one of the stuck ends releasing during the recording; DNA clipped due to photodamage; folded DNA that is clipped by photodamage; overlapping ejections.

SI Figure 7

Fig. 7. Three steps in analyzing images of ejected DNA fragments. Top: the original image captured on the camera. Middle: the result of applying the DOG filter, referred to as image C in the source code. A smoothing factor σ ≈ 0.5 mm was used. Bottom: the thresholded image, image T in the source code.

SI Figure 8

Fig. 8. Calibration for measuring ejected DNA lengths. The plot shows the observed length of the DNA in pixels as a function of its length in kbp. Histograms with a 5 pixel bin size are shown at each sample point, normalized by the total number of frames represented by each histogram. The solid curves show the calibration functions, which were manually fit to the histogrammed data.

SI Figure 9

Fig. 9. Graphs of the single ejection trajectories, showing the effect of the flow on the DNA. A four-fold change in the flow velocity appeared to have no effect on the ejection of DNA in MgSO₄ buffer. However, the data with a slower flow has an increased noise due to greater fluctuations of the DNA.

SI Figure 10

Fig. 10. Comparison of the velocity of DNA translocation at two different flow rates: 10 and 40 mL/min, corresponding to shear flows of 14 and 57 s-1, respectively. Except for several points during the middle of ejection, the velocities at both flows correspond closely, and even for the points that do not match, translocation appears faster in the slower flow. The red line indicates the position of the full-length genome, 48.5 kbp.

SI Figure 11

Fig. 11. Single trajectories from cI60 ejections in buffer A. The plot shows the amount of DNA ejected as a function of time, with trajectories aligned for visual comparison. Ejections take »4 s to reach completion in this buffer.

Movies 1-4. Real time recordings of bacteriophage lambda DNA ejection events. Recordings lasting 1,000 frames (250 s) were performed as described in the text, then single ejection events were selected from the movies and analyzed to reveal the DNA ejection process. Samples of four experimental conditions are shown. For each movie, a key presents the size scale, the elapsed time, and the frame count.

SI Movie 1

Movie 1. Phage lambda cI60 (48.5 kbp genome) in Mg buffer.

SI Movie 2

Movie 2. Phage lambda cI60 (48.5 kbp genome) in Na buffer.

SI Movie 3

Movie 3. Phage lambda cI26 b221 (38 kbp) in Mg buffer.

SI Movie 4

Movie 4. Phage lambda cI26 b221 (38 kbp) in Na buffer.

Table 1. Calibration for various flow rates and buffers

Buffer	Flow, µL/min	Shear, s−1	L₀, kbp
Mg	10	14	45
Na	10	14	25
Mg	40	57	18
Na	40	57	8

The length L₀ of DNA that stretches out to 37% of its contour length was measured as described in the text. Mg buffer had an L₀ about twice as high as Na buffer, while the flow rate also had a large effect on the value. Additionally, it was noted that other buffers containing Mg²⁺ (TM buffer, buffer A) were equivalent to Mg buffer, indicating that it is the presence of Mg²⁺ rather than the absence of Na⁺ that causes the DNA to be more flexible.