Preparation of Chemically Competent E. coli: The TSS Method

For E. coli cells to take up a foreign plasmid, they have to first be made competent - their cell walls must be weakened and made permeable to incoming DNA. This is traditionally done using divalent cations, which mask the negative charge of the phospholipid membrane, allowing the negatively-charged DNA to approach the cell without significant repulsion. The Transformation and Storage Solution (TSS) contains Mg++.

Day 1
Step	Description	Rationale
1	Prepare a primary inoculum of E. coli in 5 mL LB in a test tube.	-
2	Incubate overnight at 37°C, 170 rpm.	-
Day 2
3	Inoculate 1 mL overnight culture in 100 mL LB medium (1% inoculum)	This protocol yields one aliquot of competent cells per mL of culture. Depending on how many aliquots of competent cells you wish to make, you can vary the volume of LB medium used.
4	Incubate at 37°C, 170 rpm until the OD reaches 0.4 (~1 h 45 min for E. coli DH5α cells)	This is the early exponential phase; cells are physiologically ideal for the preparation of competent cells.
From this point, cells should always be placed in the cold (below 4°C), all buffers should be ice-cold, and all plasticware/glassware should be pre-chilled.
5	Place the culture at 4°C for 45 min	-
6	Spin down the culture at 10000 rpm, 10 min, 4°C	-
7	Resuspend the cell pellet in 1 mL ice-cold TSS buffer	-
8	Spin down the culture at 10000 rpm, 10 min, 4°C	-
9	Make 50-100 µL aliquots in chilled microfuge tubes, snap-freeze in liquid nitrogen and store at -80°C for long-term storage	-

Transformation of Chemically Competent E. coli: The Heat-Shock Method

Step	Description	Rationale
1	Thaw the competent cells on ice for 20 min	-
2	Add 1-5 µL of plasmid DNA (10 pg - 100 ng) directly into the competent cells.	-
3	Gently flick the microfuge tube to mix the cells and the DNA.	-
4	Incubate on ice for 30 min.	-
5	Heat-shock the cells at 42°C for ~45 s	The optimal heat-shock duration varies from 30 s to 90 s depending on strain, batch, and method of preparation. In our experiments, 45 s gave us a high enough transformation efficiency to proceed.
6	Place the cells on ice for 5 min	-
7	Add 950 µL SOC medium.	-
8	Incubate at 37°C, 220 rpm for 1-2 h.	The optimal incubation time varies depending on strain and antibiotic resistance marker used for selection. This step allows the few transformants to replicate and increase their plasmid number in the following generations.
9	Spread plate 100 µL of the culture on a selection plate.	-
10	Spin down the remaining culture at 5000 rpm, 10 min, resuspend in ~100 µL medium, and spread plate on a selection plate.	-
11	Incubate plates overnight at 37°C until transformant colonies are seen.	-

Isolation of Plasmid DNA from Transformants: The Miniprep Protocol

Day 1
Step	Description	Rationale
1	Inoculate one colony of the desired transformant in 5 mL of LB medium in a test tube.	-
2	Incubate overnight at 37°C, 170 rpm.	-
1	Spin down 1.5 mL of the overnight culture in a 1.5 mL microfuge tube at 5000 rpm, 10 min. Discard the supernatant. Repeat until all 5 mL has been pelleted.	-
2	Resuspend the cell pellet in 200 µL alkaline lysis solution I using a micropipette.	-
3	Keep the microfuge tube on ice for 5 min.	-
4	Add 400 µL alkaline lysis solution II along the sides of the microfuge tube.	-
5	Very gently invert the microfuge tube three times.	-
6	Keep the microfuge tube on ice for 30 s.	-
7	Add 300 µL alkaline lysis solution III.	-
8	Gently invert the microfuge tube three times.	-
9	Keep the microfuge tube on ice for 1 min.	-
10	Spin at 13000 rpm for 10 min.	-
11	Transfer the supernatant to a 2 mL microfuge tube.	-
12	Add RNase A (5 µg/mL) to the supernatant, and incubate at 65°C for 30 min.	-
13	Add an equal volume of chloroform to the microfuge tube.	-
14	Spin at 13500 rpm, 10 min, 4°C	-
15	Carefully transfer the upper aqueous layer to a new microfuge tube, avoiding white particles of protein at the water-chloroform interface.	-
16	Repeat Steps 13, 14, and 15.	-
17	Add an equal volume of ice-cold isopropanol.	-
18	Incubate on ice for 30 min.	-
19	Centrifuge at 14500 rpm, 30 min, 4°C.	-
20	Discard supernatant and add 500 µL ice-cold 70% ethanol.	-
21	Centrifuge at 14500 rpm, 30 min, 4°C.	-
22	Discard supernatant, mark the position of the pellet, and leave the excess ethanol to dry.	-
23	Resuspend the DNA pellet in 20 µL MilliQ.	-

PCR

Simple PCR

Overhang PCR

Colony PCR

DNA purification

Chloroform extraction

Gel purification

Restriction digestion

Single digestion

Double digestion

Overnight digestion

Ligation

Simple ligation

Sequential ligation

Multi-ligation

Scanning Electron Microscopy (SEM) assay

Scanning electron microscopy was done by taking samples of treated gas vesicles at various concentrations and the images were analyzed. The protocol is as follows,

Step	Description	Remarks
1	Using double sided carbon tape, stick multiple circular glass slides on a metal disc.	-
2	Load 10ul of the samples on each glass slide.	-
3	Put the disc under a vacuum desiccator until all the liquid dries up leaving only the dried gas vesicles on the slide.	If the gas vesicles are suspended in PBS, large salt crystals and salt flakes might be visible under the microscope (see image). If a large amount of gas vesicles are available, dialysis can be performed to get rid of the salt crystals.
4	Apply a gold sputter coating of 10nm thickness to the samples.	-
5	Load the disc in the Scanning electron microscope to be imaged at various magnifications.	-

[[File:T--IISc-Bangalore--SEMSalt1.png|300px]] [[File:T--IISc-Bangalore--SEMSalt2.png|300px]]
Appearance of salt in dried SEM samples

Dynamic Light Scattering (DLS) assay

Dynamic Light Scattering (DLS) sometimes also called Photon correlation spectroscopy is a technique that allows the estimation of the size of nanoparticles in a suspension by monitoring the scattering as a particle undergoes brownian motion. It proved to be extremely useful in our experiments due to the fact that it directly outputs the effective hydrodynamic size (the diameter of a hard sphere which diffuses in a similar manner as the particle of interest [4]) which is a determining factor in our model. We expect the sizes of the particles to increase after flocculation and the results can be seen in the results section of this wiki. The protocol is as simple as taking the same sample at various dilutions and putting it in a cuvette to be analyzed by the DLS machine (Zetasizer nano). The exact amounts that were required in an experiment are given next to the data that was obtained.

Spectrophotometry assay

A method to check the effective flotation was devised by using a spectrophotometer to record the OD of a dilute gas vesicle suspension at 500nm. As the gas vesicles float to the surface over time, the OD reduces giving a decreasing curve. The thermal fluctuations (read Brownian motion) at room temperature are still significant and hence the curve is not as smooth as we would expect for a heavy particle. However, the curves do provide some interesting insights into how advective and diffusive transports compete for small particle sizes. The exact protocol for flotation spectrophotometry is straightforward.

Step	Description	Remarks
1	Turn on the spectrophotometer for time analysis and set it to take readings for 2 hours at 0.1min intervals.	-
2	Add 2mL PBS to a cuvette for use as a blank.	-
3	Dilute the gas vesicle stock to 2mL to be used in the cuvette.	In all our experiments the effective gas vesicle concentration in the final suspension was ~15ng/ul.
4	Invert the cuvettes slowly a number of times and put them in their respective slots in the spectrophotometer	PBS for blank, Sample in active slot
5	Start the two hour timed run	-

Reagents

LB medium

TSS buffer

Reagent	Volume	Final concentration
2x LB medium	5 mL	50% (v/v)
25% PEG 3350 (w/v)	4 mL	10% (w/v)
DMSO	0.5 mL	5% (v/v)
2M MgCl2	0.5 mL	0.1 MgCl2
Filter sterilize the TSS buffer using a 0.22 µm filter.

Alkaline lysis solution I

Reagent	Volume	Final concentration
glucose	-	50 mM
Tris-Cl (pH 8.0)	-	25 mM
EDTA (pH 8.0)	-	10 mM
MilliQ water	-	-
Autoclave the solution at 121°C, 15 psi, 15 min to sterilize

Alkaline lysis solution II

Prepare a fresh solution just before use.
Reagent	Volume	Final concentration
1 N NaOH	2 mL	0.2 N
10% SDS (w/v)	1 mL	1% (w/v)
MilliQ water	7 mL	Up to 10 mL

Alkaline lysis solution III

Reagent	Volume	Final concentration
5 M KOAc	60 mL	3 M
glacial acetic acid	11.5 mL	11.5% (v/v)
MilliQ water	28.5 mL	Up to 100 mL
Autoclave the solution at 121°C, 15 psi, 15 min to sterilize

Chloramphenicol stock solution