Difference between revisions of "Template:Groningen/Labjournal/sfGFP(Sp)-in-pDR111-pDR111-message-plasmid"

PCR

Experiment:

10/08/16: sfGFP(Sp) was amplified from pNW plasmid together with its promoter (P_pta), 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).

PCR mixture:

25 μl PCR assay was performed according to the following protocol.

PCR set-up:

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

Amplification of the P_pta-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.

Procedure after gel validation:

DNA extraction was done according to this protocol.

Restriction digestion

Experiment:

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.

RD mixture:

20 μl RD assay was performed according to the following protocol.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

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.

Procedure after gel validation:

The upper band was cut out from the gel and DNA was extracted by Gel extraction kit (NucleoSpin® Gel and PCR Clean-up).

Ligation

Experiment:

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.

Ligation mixture:

20 μl ligation assay was performed according to the following protocol.

Transformation

Experiment:

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.

Conclusion:

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.

Validation

Experiment:

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.

RD mixture:

20 μl RD assay was performed according to the following protocol.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Sequencing:

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).

Conclusion:

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.

Experiments

Experiment:

The functionality of these constructs was checked by integration into the B. subtilis genome 168 trp+ and imaging under the microscope.

Integration into the genome of B. subtilis 168 trp+:

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 P_pta-sfGFP(Sp)-3TER, which is located between amyE front flanking region and amyE back flanking region.

Experiment set-up:

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.

Microscopy experiment set-up:

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.

Conclusion:

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.