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Cell Protocols
Molecular Biology
Assays
Reagents

Cell-Based Protocols

Preparation of competent cells — 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.	-

Molecular Biology

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

Transfer all the mention components to the PCR eppendorfs. Gently spin them down to ensure that all the components have properly mixed.

Component	25 µl Reaction	50 µl Reaction	Final concentration
10x Standard Taq Reaction buffer	2.5 µl	5 µl	1x
10mM dNTPs	0.5 µl	1 µl	200uM
10um Forward Primer	0.5µl	1µl	0.2µM (0.05-1µM)
10 µM reverse Primer	0.5µl	1µl	0.2µm (0.05-1µM)
Template DNA	variable	variable	<1000ng
Taq DNA Polymerase	0.125µl	0.25µl	1.25 units/50µl PCR Reaction
Nuclease-free water	to 25µl	to 50µl

Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling

Thermocycling conditions for routine PCR

Step	Temp	Time
Intial denaturation	95 degrees	30 seconds
30 cycles	95 degrees 45-68 degrees 68 degrees	15-30 seconds 15-60 seconds 1 minute/kB
Final extension	68 degrees	5 minutes
Hold	4-10 degrees

Colony PCR

Transfer all the mention components to the PCR eppendorfs. Gently spin them down to ensure that all the components have properly mixed.

Component	25 µl Reaction	50 µl Reaction	Final concentration
10x Standard Taq Reaction buffer	2.5 µl	5 µl	1x
10mM dNTPs	0.5 µl	1 µl	200µM
10um Forward Primer	0.5µl	1µl	0.2µM (0.05-1µM)
10 µM reverse Primer	0.5µl	1µl	0.2µM (0.05-1µM)
Taq DNA Polymerase	0.2µl	0.4µl
Water + colony	19.3µl	38.6µl

Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling

Thermocycling conditions for colony PCR

Step	Temp	Time
Intial denaturation	95 degrees	30 seconds
30 cycles	95 degrees 45-68 degrees 68 degrees	15-30 seconds 15-60 seconds 1 minute/kB
Final extension	68 degrees	5 minutes
Hold	4-10 degrees

DNA purification

Chloroform extraction

Gel purification

Step	Procedure	Rationale
1.	Excise the DNA band of interest using an ethanol cleaned razor blade or a scalpel on a transilluminator	Minimize gel volume by cutting as small a slice as possible. Use of long wave transilluminator and short handling time will lead to better quality DNA
2.	Weigh the gel slice in a microcentrifuge tube. Add 3 volumes (µl) of buffer GB to 1 volume (mg) of gel
3.	Indubate at 50 degree Celcius until the agarose gel is completely melted (5-10 min)	To help effective dissolving of the gel, vortex every 2-3 minutes during incubation
4.	AFter the slice has dissolved completely, check that the colour of the mixture is yellow (similar to buffer GB)	If the colour of the mixture becomes brown or purple, add 10ul of 3M Sodium Acetate, pH 5.0 and mix. The colour of the mixture will turn to yellow
5.	(Optional) Add 1 gel volume of the isopropanol to the sample and vortex to mix	Do not centrifuge at this step
6.	Transfer the mixture to a SV column. Centrifuge for 1 min at 10,000x g and above (>12,000 RPM). Discard the pass through and res-insert the SV column intro the collection tube	IF the mixture volume is greater than 700µl, apply the mixture twice; apply 700ul of the mixture, dpin down, discard the pass through,re-insert empty collection tube, and repeat until all the mixture has been applied to the SV column
7.	(Optional) Apply 500µl of buffer GB to the SV column. Centrifuge for 30 seconds. Discard the passthrough and re-insert the SV column into the collection tube.	This is to further remove any traces of agarose and required only for direct use of DNA in very sensitive applications
8.	Add 700µl of buffer NW to the SV column. Centrifuge for 30 seconds. Discard the pass-through. Reinsert the SV column into the collection tube	If the purified DNA will be used for salt sensitive applications, let the SV column stand for 5 min after addition of buffer NW, making some amount of wash buffer flow through the column by gravity before centrifugation
9.	Centrifuge for an additional 1 min to remove residual wash buffer. Transfer the SV column to a new 1.5 ml centrifuge tube.	If residual ethanol still remains, centrifuge again for 1 min at full speedbefore transferring to a new 1.5 ml microcentrifuge tube
10.	Apply 50µl of buffer EB or ddH2O to the center of the membrane in the SV column, let stand for 1 min and centrifuge for 1 min	Ensure that buffer EB or distilled water is dispensed directly onto the centre of the SV column for optimal elution of DNA. For larger fragment (>5 kB), used pre-warmed elution buffer (70 degree celcius) for best efficiency. For long term storage, eluting in buffer EB (10 mM Tris Cl, pH 8.5 or TE, pH 8.0 and storing under -20 degree celcius is recommended.

Restriction digestion

Step	Procedure	Rationale
1.	Select the Restriction enzymes you need to digest your plasmid with.
2.	Select the appropriate buffer for the working of the enzyme.	If you are doing double digest, then you need to find a buffer which gives optimal activity with both the enzymes. If you are not able to find any such buffer, then you have to proceed with sequential digestion.
3.	A typical restriction digestion reaction could look like this: 1 µg DNA 1 µl each of Restriction enzymes 5 µl 10x buffer 3 µl 10x BSA(if recommended, usually most of the enzymes nowadayshave BSA in them) x µl dH2O (bring it up to 50µl)
4.	MIx well by gently pipetting
5.	Incubate at appropriate temperature (usually 37 °C) for 1 hour.	You can also do an overnight digestion by incubating overnight, just add less amount of enzyme.
6.	After the digestion, run a 1% agarose gel to check if the digestion has worked.

One can also proceed to gel elution to get the specific band required.
To purify the plasmid for further usage perform Chloroform extraction, since the efficiency of Gel elution is very low.
For sequential digestion, if need to be done, heat inactivate the enzyme by incubating at 70°C for 10-15min
If your enzyme did not cut, check to make sure that it isn’t methylation sensitive. Plasmids grown in Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction sites.

Ligation

The volume of vector DNA and insert DNA used in the ligation will vary depending on the size of each and their concentration. However, for most standard cloning (where the insert is smaller than the vector) a 3 (insert):1 (vector) ratio will work just fine

Step	Procedure
1.	Combine the following in a PCR or an Eppendorf tube: 25ng Vector DNA 75ng Insert DNA Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer) 0.5-1μL T4 DNA Ligase H20 to a total of 10μL
2.	Incubate at room temperature for 2 hrs or overnight at 16°C>
3.	Proceed with bacterial transformation. You can use 10 µL of ligation mixture with 100 uL of competent cells. There is no need to purify the ligation mixture.

Running the controls

Control	Ligase	Interpretation
Uncut Vector	-	Checks viability of competent cells and verifies the antibiotic resistance fo the plasmid.
Cut Vector	-	Background due to uncut vector
Cut Vector	+	Background due to vector recircularization - most useful for phosphatase treated vector

Our Assays

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 10µl 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.	-

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) 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 (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

Reagent	Volume	Final concentration
Prepare a fresh solution just before use.
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

@@ Line 717: / Line 717: @@
 <h2>Spectrophotometry assay</h2>
-<p>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.
+<p>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 (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.</p>
 <table>

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