Template:Groningen/Labjournal/sfGFP(Sp)-in-pDR111-pDR111-message-plasmid
sfGFP(Sp) is a reporter gene originally optimized for Streptococcus pneumoniae. It has been shown when expressed in B. subtilis that the signal is even brighter than the one from sfGFP(Bs) (Overkamp et al. 2013). sfGFP(Sp) was cloned either only to pDR111 integration plasmid or to
pDR111+message plasmid. These plasmids were constructed with an interest to
have easier screening when integrated into the B. subtilis genome. pDR111 is an integration plasmid which can be integrated into the B.
subtilis genome. By double cross-over it replaces amyE gene, which is
necessary for production of alpha-amylase,
with desired insert which is located between amyE front flanking region and
amyE back flanking region. See the plasmid map below. 10/08/16: sfGFP(Sp) was amplified from pNW plasmid together with its
promoter (Ppta), which is a constitutive promoter from Geobacillus
DSM2542 for phosphate acetyl transferase expression, and a triple
terminator (3TER). Primers used for the amplification were F-gfp insert
and R-gfp insert (primer sequences can be found here). 25 μl PCR assay was performed according to the following protocol. For detailed information on how to prepare and run agarose gel
see following protocol. Amplification of the Ppta-sfGFP(Sp)-3TER was successful. It was
verified by DNA electrophoresis. Correct size of a band
could be seen and that was 1147 bp. However second band could be seen,
that was probably caused by unspecific binding of the primers used.
Therefore lower band of correct size was cut out from the gel and DNA
was extracted. DNA extraction was done according to this protocol. 10/08/16: On this day restriction digestion of PCR product of the
sfGFP(Sp), pDR111 integration plasmid and pDR111+message
integration plasmid was done. sfGFP(Sp) as an insert was cut with BglII
and NheI restriction enzymes. Integration plasmid pDR111 and
pDR111+message as a vector was cut with BamHI and NheI. BglII could not
be used as a restriction enzyme for pDR111 due to multiple recognition
site of this cutter. BamHI as a compatible restriction enzyme to BglII
was used instead. 20 μl RD assay was performed according to the following protocol. For detailed information on how to prepare and run agarose gel
see following protocol. RD of the PCR product of sfGFP(Sp), integration plasmid pDR111 and
pDR111+message was successful. RD of pDR111 and pDR111+message was
verified by DNA electrophoresis. Correct sizes of bands could
be seen and that was 6479 bp and 7034 bp, respectively. The upper band was cut out from the gel and DNA was extracted by Gel
extraction kit (NucleoSpin® Gel and PCR Clean-up). 10/08/16: Restriction digestion was followed by ligation at
room temperature for 45 min. Vector (integration plasmid pDR111 and
pDR111+message) and insert (sfGFP(Sp)) was ligated in 1:5 molar ratio. 20 μl ligation assay was performed according to the following
protocol. 10/08/16: Ligation was followed by transformation into E. coli Top10.
Selection was made on 100 μg/ml ampicillin LB agar plates.
Transformation protocol used can be found here. 11/08/16: Next day we could assume that our transformation was
successful -> colonies were obtained on the plates of E. coli Top10
strain. To verify correct transformants we grew some of the colonies
overnight in 3 ml LB with 100 μg/ml ampicillin (see cell culture
protocol). On the next day plasmid
purification and restriction digestion control was done. Transformation of pDR111+sfGFP(Sp) and pDR111+message+sfGFP(Sp) into
E. coli Top10 was assumed to be successful. To see if we obtained
correct clones we did a restriction digestion control with EcoRI
restriction enzyme. 12/08/16: Next day plasmid purification was done according to this
protocol and restriction digestion control with EcoRI
restriction enzyme was done to see if correct clones were obtained.
This enzyme has 2 recognition sites in our construct. Therefore two
bands (1535 bp and 6081 bp from pDR111+sfGFP(Sp) and 2090 bp and 6081
bp from pDR111+message+sfGFP(Sp)) should have been obtained on the
agarose gel. Moreover we sent this new constructed plasmid for
sequencing. 20 μl RD assay was performed according to the following protocol. For detailed information on how to prepare and run agarose gel
see following protocol. Plasmid pDR111+sfGFP(Sp) and pDR111+message+sfGFP(Sp) was sent for
sequencing. Primers used for
the sequencing were F-gfp insert for pDR111+sfGFP(Sp) and F-gfp insert
and F-message sequence for pDR111+message+sfGFP(Sp) (primer sequences
can be found here). Figure 5 shows two correct bands (1535 bp and 6081 bp) when the
pDR111+sfGFP(Sp) was cut. Figure 6 shows two correct bands (2090 bp
and 6081 bp) when the pDR111+message+sfGFP(Sp) was cut. Therefore we
assumed that our cloning was successful and correct plasmids were
obtained. Sequencing results confirmed our assumption as you can see in
Figure 7, 8 and 9. The functionality of these constructs was checked by integration
into the B. subtilis genome 168 trp+ and imaging under the microscope. sfGFP(Sp) in pDR111 and pDR111+message was integrated into the B.
subtilis 168 trp+ genome. pDR111 is an integration plasmid which can be
integrated into the B. subtilis genome. By double cross-over it
replaces amyE gene, with desired insert, in this case insert is
Ppta-sfGFP(Sp)-3TER, which is located between amyE front
flanking region and amyE back flanking region. 10 μl of sfGFP(Sp) in pDR111 and pDR111+message plasmids were
transformed into the B. subtilis 168 trp+ strain (see transformation
protocol). Selection was made on 150 μg/ml
spectinomycin LB agar plates. B. subtilis cells were grown overnight in 3 ml LB with 150 μg/ml
spectinomycin at 37°C and 220 rpm. On the next day microscopy slide was
prepared as explained in the following protocol. Cells
were imaged using phase contrast: filter POL 50% 1 s exposure, green
fuorescence: FITC, 0.8 s exposure, objective: Olympus 100X/1.40,
camera: CoolSNAP_HQ/HQ2-ICX285 and software: Resolve3D softWoRx-Acquire
version. Integration into the B. subtilis 168 trp+ strain of the plasmids
with the reporter sfGFP(Sp) was successful. Multiple colonies were
obtained on selection plates. B. subtilis cells were subsequently grown
and checked under the microscope to see if there is an expression of
GFP (see Figure 10). We could definitely see the GFP signal when
observing the cells under the microscope. This was big enough proof
that our plasmid construction followed by integration into the B.
subtilis genome was successful.sfGFP(Sp) in pDR111 and in pDR111+message plasmid
PCR
Experiment:
PCR mixture:
PCR set-up:
95ºC 2:00 min 95ºC 30s (30X) 57ºC 30s (30X) 72ºC 1:30 min (30X) 72ºC 2:00 min 10ºC on hold DNA Electrophoresis:
Conclusion:
Procedure after gel validation:
Restriction digestion
Experiment:
RD mixture:
DNA Electrophoresis:
Conclusion:
Procedure after gel validation:
Ligation
Experiment:
Ligation mixture:
Transformation
Experiment:
Conclusion:
Validation
Experiment:
RD mixture:
DNA Electrophoresis:
Sequencing:
Conclusion:
Experiments
Experiment:
Integration into the genome of B. subtilis 168 trp+:
Experiment set-up:
Microscopy experiment set-up:
Conclusion:
