Template:Groningen/Labjournal/sfGFP(Sp)-in-pSB1C3

sfGFP(Sp) in pSB1C3 (BBa_K1930006)

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). To submit sfGFP(Sp) reporter gene as BioBrick (BBa_K1930006) we cloned it in the pSB1C3 standard iGEM backbone.

PCR

Experiment:

06/10/16: sfGFP(Sp) was amplified from the pDR111+sfGFP(Sp) plasmid, that we constructed during the project, with the primers prefix sfGFP(Sp) and suffix sfGFP(Sp) (primer sequences can be found here).

PCR mixture:

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

DNA Electrophoresis:

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

Figure 1. DNA electrophoresis with amplified sfGFP(Sp) gene (763 bp in size).
Conclusion:

The sfGFP(Sp) was successfully amplified from the pDR111+sfGFP(Sp) plasmid.

Procedure after gel validation:

PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience.

Restriction digestion

Experiment:

The sfGFP(Sp) was cut with EcoRI and PstI. The backbone pSB1C3 (BBa_J04450) was digested with the same enzymes. This construct is carrying RFP reporter therefore it was used for easier screening after transformation. You could see self-ligations as red colonies and the correct ones as white ones.

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.

Figure 2. DNA electrophoresis of EcoRI and PstI digested pSB1C3 - BBa_J04450. The RFP insert is 1069bp and the pSB1C3 backbone is 2019 bp.
Conclusion:

The digestion was successful because bands for both expected fragments could be seen on the gel, namely RFP insert is 1069bp and the pSB1C3 backbone is 2019 bp.

Procedure after gel validation:

The upper band of 2000 bp was cut out from the gel and DNA was extracted with Agarose Gel Extraction Kit – Jena Bioscience. The PCR product was not checked on the gel after the digestion but immediately cleaned up with NucleoSpin® Gel and PCR Clean-up.

Ligation

Experiment:

The cut and cleaned sfGFP(Sp) was ligated to the cut and cleaned pSB1C3 in a ratio of 2:1.

Ligation mixture:

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

Transformation

Experiment:

07/10/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the transformation protocol. Cells were plated on 50 μg/ml chloramphenicol LB agar to select the correct construct.

09/10/16: Colonies were picked to perform colony PCR to find the correct constructs with the primers prefix sfGFP(Sp) and suffix sfGFP(Sp). Find primer sequences here.

Figure 3. Top10 E. coli colonies on 50 μg/ml chloramphenicol LB agar plates after transformation with sfGFP(Sp) in pSB1C3.
PCR mixture:

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

PCR set-up:
95ºC2:00 min
95ºC30s(30X)
60ºC30s(30X)
72ºC1:30 min(30X)
72ºC2:00 min
10ºC on hold
DNA electrophoresis:

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

Figure 4. DNA electrophoresis of the colony PCR product. C - is the water control. Samples 1-5 are from colony 1-5.
Conclusion:

All 5 samples show the right band by 763 bp. Therefore all of them were grown overnight to harvest the plasmid the following day.

Validation

Experiment:

10/10/16: Grown cultures of E. coli Top10 with the construct sfGFP(Sp) in pSB1C3 were used to obtain (glycerol stocks) and plasmid isolation was performed with QIAprep® Spin Miniprep Kit. Some of the overnight cultures seemed to already express the sfGFP (see Figure 5). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Figure 5. Pellet of the overnight cultures from sample 2 and 5. Looks like the sfGFP(Sp) is also expressed in E. coli Top10.
Sequencing:

Sequencing results showed that sfGFP(Sp) gene is present (see Figure 6). However something happened with the prefix and the suffix most probably during cloning steps. It looks that prefix and also suffix was disrupted by insertion of few base pairs. We cannot really explain what happened and due to time limitation we could not fix this problem. However sfGFP(Sp) is clearly expressed in E. coli (see Figure 5).

Figure 6. Sequencing result of sfGFP(Sp) in pSB1C3. GFP gene itself is underlined by red line.
Conclusion:

The sfGFP(Sp) was cloned to the backbone pSB1C3. However from the sequencing results we could see that this part contains bad prefix and suffix (see Figure 6). GFP gene is underlined by red line on Figure 6, however you could also see the bad prefix and suffix.

Experiments

See decoy experiments with sfGFP(Sp) in B. subtilis.