Template:Groningen/Labjournal/Key-in-pSB1C3

Key sequence in pSB1C3 (BBa_K1930000)

The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The key sequence was created by our software. It is therefore artificial DNA. We ordered the encryption key sequence as gBlock from IDT (Integrated DNA technologies). To submit our key sequence as BioBrick (BBa_K1930000) we cloned it in the pSB1C3 standard iGEM backbone.

PCR

Experiment:

27/09/16: The key was PCR amplified (see following protocol) from the pDR111+key plasmid with the primers key prefix and key suffix (see primer list). The correct size 187 bp of the product was checked by DNA electrophoresis. The PCR product was stored at 4°C.

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 gels see following protocol.

Figure 1. DNA electrophoresis of amplified key sequence (187 bp in size).
Conclusion:

The key was successfully amplified with prefix and suffix from the pDR111+key plasmid.

Procedure after gel validation:

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

Restriction digestion

Experiment:

28/09/16: The PCR product of the key sequence 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:

30 μl RD assay was performed according to the following see following protocol.

DNA Electrophoresis:

The digestion mixture of backbone pSB1C3 - BBa_J04450 was loaded on a gel to extract the digested backbone.

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

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

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

Procedure after gel validation:

The upper band of 2019 bp was cut out from the gel and DNA was extracted by (Agarose Gel Extraction Kit – Jena Bioscience). The PCR product of the key sequence was not checked on the gel after the digestion but immediately cleaned up with (NucleoSpin® Gel and PCR Clean-up). The concentration of the digested and cleaned key sequence was measured with the Nanodrop, 10,3 ng/μl were obtained.

Ligation

Experiment:

07/10/16: The EcoRI, PstI cut pSB1C3 backbone was ligated with the EcoRI, PstI cut key.

Ligation mixture:

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

Transformation

Experiment:

08/10/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the protocol. Cells were plated on 50 μg/ml chloramphenicol LB agar to select for the correct constructs. The next day colonies were picked to perform colony PCR to find the correct constructs with the primers key only prefix and key only suffix. Find primers here.

Figure 3. E. coli Top10 cells transformed with key 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 gels see the following protocol.

Figure 4. Result of the colony PCR. Samples from colony 1-9 are showing the correct size of the key sequence which is 187 bp. C - is the water control.
Conclusion:

The transformation of the key sequence in pSB1C3 to E. coli Top10 was successful. We have obtained correct insert size when colony PCR was done. And that was 187 bp.

Validation

Experiment:

09/10/16: Grown cultures of E. coli Top10 with the construct key in pSB1C3 were used to obtain glycerol stocks and plasmid isolation was performed (Fast-n-Easy Plasmid Mini-Prep Kit - Jena Bioscience). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Sequencing:

The BioBrick key in pSB1C3 (BBa_K1930000) from colonies 1 and 3 was sent for sequencing with the primers VF2 and VR, (see primer list).

Figure 5. Sequencing result for the key in pSB1C3 (BBa_K1930000) from colony 1 with VF2.
Figure 6. Sequencing result for the key in pSB1C3 (BBa_K1930000) from colony 1 with VR.
Conclusion:

The sequencing result proofed the successful integration of the key into the pSB1C3. First BioBrick was made!

Experiments

Experiments:

See integration of the key sequence in B. subtilis via the BioBrick BBa_K823023 integration plasmid in Proof of concept experiment.