Template:Groningen/Labjournal/improved-nucA-in-pSB1C3

Improvement of BBa_K729004

In our project we wanted to construct system which will destroy DNA sequence if tetracycline or tetracycline analog ATc (anhydrotetracycline) will not be added when growing cells. For this construct four parts were needed. The main part nuclease from Staphylococcus aureus (nucA) was ordered from iGEM headquarters as an available BioBrick BBa_K729004 from iGEM team University College London 2012. We have tried to assemble our desired construct (see first step), but after sequencing of this plasmid (RBS+nucA in pSB1C3), we have realized that the nuclease BioBrick BBa_K729004 has incorrect prefix. Due to time limitation it was decided that we will not continue with cloning of our desired nuclease construct, but we will make an improvement of this part and we will fix incorrect prefix in this part.

PCR

Experiment:

12/10/16: nucA was amplified from BBa_K729004 with nucA+prefix and nucA+suffix primers (primer sequences can be found here).

PCR mixture:

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

PCR set-up:
95ºC2:00 min
95ºC30s(30X)
58ºC30s(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 1. DNA electrophoresis with amplified NucA (602 bp in size).
Conclusion:

Amplification of the nucA and addition of the prefix and suffix to this sequence was successful. It was verified by DNA electrophoresis. Correct size of a band could be seen and that was 602 bp.

Procedure after gel validation:

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

Restriction digestion

Experiment:

13/10/16: On this day restriction digestion of PCR product of the nucA and pSB1C3 (BBa_J04450) was done. NucA as an insert was cut with EcoRI and PstI restriction enzymes. pSB1C3 (BBa_J04450) as a vector was cut with exactly same restriction enzymes.

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 with digested nucA with EcoRI and PstI.
Figure 3. DNA electrophoresis with digested pSB1C3 (BBa_J04450) with EcoRI and PstI.
Conclusion:

RD of the PCR product of the nucA and pSB1C3 (BBa_J04450) was successful. RD of both was verified by DNA electrophoresis. Correct size of bands could be seen and that was ∼600 bp (nucA) and 2019 bp (pSB1C3).

Procedure after gel validation:

Bands with cut nucA and the upper band with cut pSB1C3 were cut out from the gel and DNA was extracted by Gel extraction kit(NucleoSpin® Gel and PCR Clean-up).

Ligation

Experiment:

13/10/16: Restriction digestion was followed by ligation at room temperature for 1 hour. Vector (pSB1C3) and insert (nucA) was ligated in 4:12 and 8:12 molar ratio.

Ligation mixture:

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

Transformation

Experiment:

13/10/16: Ligation was followed by transformation into E. coli Top 10 cells. Selection was made on 50 μg/ml chloramphenicol LB agar plates. Transformation protocol used can be found here.

14/10/16: Next day we could assume that our transformation was successful -> colonies were obtained on the plates of E. coli Top 10 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. Moreover samples were sent for sequencing.

Figure 4. Transformation of nucA in pSB1C3.
Conclusion:

Transformation of improved nucA in pSB1C3 into E. coli Top 10 was assumed to be successful. To see if we obtained correct clones we did a restriction digestion control with EcoRI and PstI restriction enzymes and we sent the samples for sequencing.