TITLE: Fluorescent labeling Bacillus mycoides
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>GFP method is performed in bacteria to localize a desired peptide in the bacteria. In this experiment, we are focusing on the Nlp-14a. In the paper written by </span><span style = "font-style:italic;">(Yi,2017) </span><span>GFP was mutated on a B. mycoides E18 strain to study the plant-interaction studies. In their paper, they constructed a superfolder green fluorescent protein (sfGFP) and red fluorescent protein (mKate2) in the B. mycoides E18 strain. They tracked the GFP- tagged bacterial strain after inoculating in Chinese cabbage plants in a hydroponic system. They noticed that the bacterial strain rapidly attached to the plant during colonization and formed a matrix. </span></div></div>

[STEPS]
?. [Preparation of B. mycoides cells for GFP]
Prepare the B. mycoides strain aliquotes for electroporation.
?. [Preparation of B. mycoides cells for GFP]
Cultivate the bacterial strain overnight in LB broth at  and at 180 rpm.
30 °C
?. [Preparation of B. mycoides cells for GFP]
Transfer  of the overnight culture into  of LB medium (with 2% [wt/wt] glycine) and incubate it at   and 180rpm until optical density at 600 nm is 0.4 to 0.7.
1 mL
100 mL
30 °C
?. [Preparation of B. mycoides cells for GFP]
Centrifuge the cells and wash the pellets with increasing concentrations of ice-cold glycerol (2.5%, 5%, and 10%). Resuspend this pellet in precooled electroporation buffer (10% glycerol) and shock freeze in liquid nitrogen.
?. [Preparation of B. mycoides cells for GFP]
Add the library vector DNA in an amount of   to the cells, and perform electroporation. The settings for electroporation are 2.0 kV, 25 lF and 200 X in a 2-mm cuvette using a Bio Rad Gen Pulser II electroporation system (Bio-Rad).
2 µg
?. [Preparation of B. mycoides cells for GFP]
Add  of LB medium and grow the cells for  at  and 150 rpm for recovery and then plate on LB-Cm4 agar.
1 mL
30 °C
?. [Preparation of B. mycoides cells for GFP]
After   of growth at , harvest the colonies from the plates and pool in LB medium
30 °C
?. [Preparation of B. mycoides cells for GFP]
Store the libraries at  as 15% glycerol stocks.
-80 °C
?. [FACS]
Inoculate the B. mycoides strain mKate2mut library in  of LB-Cm4 and grow at  or  to an OD600nm of 0.3-0.6.
50 mL
?. [FACS]
B. mycoides has been seen to show extensive cell-chaining and hence a mild sonication step of 4 rounds of 3 X 10 pulses of 1s with an amplitude of 30% can be applied to disassemble the aggregated cells.
?. [FACS]
Sort the cells on a flow cytometer at 20 psi using a  nozzle at a flow rate of 1.0 with the highest sort precision mode (0– 32-0 sort purity mask).
?. [FACS]
Using a sequential gating strategy with FCS height versus widths, followed by SCC height versus width, cellular debris, and chained cells can be excluded.
?. [Screening of FP variants and flow cytometry measurements]
After FACS sorting, plate the final fluids containing bright cells on LB-Cm4 plates and grown them overnight at .
30 °C
?. [FACS]
To separate the brightest variants choose a cutoff of 3% of the brightest event in the first round of cell sorting and 0.3% of the brightest events in the second round of sorting with the light scatter parameters.
?. [Strains and Growth conditions of bacteria]
Routinely culture Bacillus strains in Luria-Bertani (LB) medium at   with aeration at 200 rpm.
30 °C
?. [Screening of FP variants and flow cytometry measurements]
Observe the colonies using a fluorescence microscope. Keep the filter setting for GFP as excitation at 460/480nm and emission at 495/540 nm with a 485 nm dichromatic mirror; for RFP, the filter setting can be kept as excitation at 545/580 nm and emission at  610 nm with a 600 nm dichromatic mirror.
?. [Screening of FP variants and flow cytometry measurements]
Calculate the total cell fluorescence the formula is: Corrected total cell fluorescence (CTCF) = Integrated Density – (Area of selected cell x Mean fluorescence of background readings)
?. [Screening of FP variants and flow cytometry measurements]
Capture the images on a camera and calculate the intensity of single-cell with Image J software.
?. [Strains and Growth conditions of bacteria]