DNA assembly of [RCF10 / RCF25] BioBricks based on NEB protocols
Protocol to assemble a composite part designated into a vector backbone.
A. Digestion
Digest upstream BioBrick with EcoRI-HF® and SpeI. For translational fusions, EcoRI-HF and AgeI were used.
Upstream Part
1 µg
EcoRI-HF
1 µl
Spel or AgeI
1 µl
10X NEBuffer 2.1
5 µl
H2O
ad 50 µl
Digest downstream BioBrick with XbaI and PstI. For translational fusions, NgoMIV and PstI were used.
Upstream Part Plasmid
1 µg
Xbal or NgoMIV
1 µl
Pstl
1 µl
10X NEBuffer 2.1
5 µl
H2O
ad 50 µl
Digest the vector EcoRI-HF® and PstI.
Destination Plasmid DNA
1-4 µg
EcoRI-HF
1 µl
Pstl
1 µl
10X NEBuffer 2.1
5 µl
H2O
ad 50 µl
Incubate all three restriction digest reactions at 37°C for 1 h. 1 µl of Quick CIP was added to the backbone restriction reaction for dephosphorylation.
Digested products were checked by gel electrophoresis and subsequently purified from the gel. Inserts were purified by using the PCR clean-up Kit (Macherey-Nagel / Promega).
B. Ligation
For ligation of two BioBricks into a designated backbone, the following procedure was performed:
Part 1 digestion
1:3 (Vector:insert ratio)
Part 2 digestion
1:3 (Vector:insert ratio)
Destination Plasmid digestion
100 ng
10X T4 DNA Ligase Buffer
2 µl
T4 DNA Ligase
1 µl
H2O
ad 20 µl
Incubate at room temperature for 30 min and then heat inactivate at 80°C for 20 min. Purify using PCR clean-up kit and transform into an appropriate recipient.
Transformation via electroporation
1. Production and preparation of recipient (e.g. Dh10alpha E. coli)
Main culture was started using a 1:100 overnight culture:main culture (v/v) and incubated at 37°C until OD600 of ∼ 0.6 was reached. Cells were harvested via centrifugation for 5 min at 10.000 xg and washed two times in 25 mL ice-cold MQ-H2O. After washing, cells were resuspended in 200 µl MQ-H2O and placed on ice.
2. Electroporation and confirmation
5 µl linear DNA or 1 µl plasmid DNA was mixed with 50 µl competent cells and transferred into ice-cold electroporation cuvettes. Samples were electroporated and immediately transferred into 1 ml LB and incubated for 1 h at 37°C. Mixture was spread onto appropriate (dependent on backbone applied) antibiotic LB plates followed by overnight incubation at 37°C. The next day, plates were restreaked to gain single colonies.
Allelic replacement using lambda-RED and tetRA replacement
This protocol, written by Joyce Karlinsey, has been optimized for Salmonella Typhimurium LT2 in the lab of Dr. Kelly T. Hughes. Cite: Genetic Transplantation: Salmonella enterica Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in Genetically Intractable Systems, Karlinsey, Hughes.
Measuring expression capacity of minicells
For the characterization of the expression capacity of minicells, the fluorescence of mCherry was measured over time. Minicells were purified from a strain that constitutively expressed mCherry and from a control strain which did not express mCherry. 100 µl of the purified minicells were distributed into a 96 well plate. As a control, translation of mCherry was inhibited by the addition of tetracycline to a final concentration of 30µg/ml. For each control, as well as for the mCherry expressing minicells, the analysis was performed in three different technical replicates. Fluorescence was measured every 5 minutes for 6h at 37°C under constant shaking. mCherry excitation was performed at 590 nm excitation, 9 nm bandwidth; emission was measured at 620 nm, 20 nm bandwidth.
PCR
Amplification of Inserts for Cloning and Cassettes for Homologous Recombination
A Q5 high fidelity polymerase from New England Biolabs was used for this purpose, as it has a very low error rate even with large amplifications. The procedure is shown in the table below.
Component
50 µl reaction
Q5 High-Fidelity 2x Master Mix
25 µl
10 µM forward primer
2.5 µl
10 µM reverse primer
2.5 µl
Template DNA
variable
Nuclease-free H2O
ad 50 µl
Initial Denaturation
98°C
30s
35 cycles
98°C
10s
NEB Tm Calculator
30s
72°C
30s/kb
Final Extension
72°C
2 min
Hold
4°C
∞
Colony PCR
For colony PCR, lab internal manufactured Taq polymerase was used. Instead of the isolated plasmid to be amplified, some cell material of a transformant colony was used as a template, from which the plasmid to be amplified was released by prolonged initial denaturation.
Master Mix [µl]
Stock
1x
1x PCR buffer
5x
4.0
1.5 mM MgCl2
25 mM
1.2
800 µM dNTP’s
20 mM
0.8
0.5 µM primer 1
10 µM
1.0
0.5 µM primer 2
10 µM
1.0
Go-Taq Polymerase (U)
5 U/µl
0.2
Template DNA
Picked colony
Nuclease-free H2O
ad to 20 µl
Cycle
Time
Temperature
initial denaturation
3 min
95 °C
denaturation
5-15 sec
95 °C
annealing
30 sec
Ann
35 cycles
extension
60 sec/1kb
72 °C
final extension
5 min
72 °C
soak
∞
12 °C
Agarose Gel Electrophoresis
To separate DNA based on size, gel electrophoresis was applied to agarose at a concentration of 1% (depending on desired fragment size) [w/v]. The 100 bp extended ladder from ROTH was used to determine fragment size. Separation was performed at 100 V for 25 - 45 min, and the gel was placed in a 0.1% [w/v] GelRed solution bath for DNA staining. GelRed intercalates in DNA, which makes DNA visible under UV light.
Isolation of Plasmid DNA
A total of 4 ml of the overnight culture was used to obtain plasmid DNA using the “Monarch® Plasmid Miniprep Kit“ from NEB.
Microscopy - time-lapse
An overnight culture of a minicell producing strain, which expressess mCherry from a constitutive promoter was diluted 1:100 in LB medium and grown at 37 °C until the late log phase was reached. The culture was diluted to an OD600 of 0.1 and 2.5 µl were spread on an agarose pad in a Gene Frame (Thermofisher scientific). Cells were visualized every 15 min for a period of 5 h. To image mCherry a 640 nm laser set to 5% was used. Samples were exposed to the laser for 100 ms.
Microscopy - PMF Measurement
Minicells expressing GFP under regulation of the lldR promoter were purified according to the protocol “purification of minicells”. Purified minicells were sedimented at 20,000 xg for 1 min and resuspended in 600 µl LB medium. The resuspended minicells were incubated at 37 °C, 700 rpm. At each given time point indicated: 50 µl of the minicell sample were taken and mixed with 450 µl 1x PBS, supplemented with 0.2% glucose.
3,3'-Dipropylthiadicarbocyanine Iodide (DiSC3(5)) was added to the culture to a final concentration of 0.2 mM and incubated for 5 min at 37 °C under constant shaking. A sample of the culture was then spread on an agarose pad in a gene frame (Thermo Fisher scientific) and imaged. To image GFP and DiSC3(5), a 488 nm and a 640 nm laser set to 5% intensity were used. GFP was imaged using an exposure time of 100 ms and DiSC3(5) of 200 ms.
Microscopy - Cell Membrane Staining and High Resolution Microscopy
FM 4-64 was added to a final concentration of 5µg/mL to a sample of either purified minicells or a growing culture of minicell-producing strain. The sample was then incubated for 30 min at 37 °C and 700 rpm. Subsequently, the minicells were centrifuged at 20,000 xg, for 1 min and the supernatant was discarded. The pellet was resuspended in 1x PBS of the initial volume. Cells were then imaged using the STED microscope from Abberior.
Characterization of Promoter Activity
For characterization of promoter activity, bacteria expressing GFP from the lldR promoter were grown to early log phase and subsequently diluted to an OD600 of 0.01. 100 µl aliquots of the cell culture were then distributed in a flat bottom black plate (Greiner) in triplicates for each lactic acid concentration. Plate was then incubated for 1 h at 37 °C under constant shaking. After 1 h incubation, expression of GFP was induced by addition of lactic acid to different concentrations ( 0 mM, 0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM, 3.2 mM, 6.4 mM, 12.5 mM, 25 mM and 50 mM). Fluorescence was then measured every 15 min for a period of 14 h. GFP excitation was performed at a wavelength of 488 nm and emission was measured at 522 nm. Relative fluorescence units were calculated by dividing the average fluorescence by the OD600.
