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 message sequence was created by our software. It is therefore artificial DNA. We ordered the encryption message sequence as gBlock from IDT (Integrated DNA technologies). To submit our message sequence as BioBrick (BBa_K1930001) we cloned it in the pSB1C3 standard iGEM backbone.
PCR
Experiment:
06/10/16: The message sequence was amplified from pDR111+message plasmid.
Primers used for the amplification were message prefix and message
suffix (primer sequences can be found here).
PCR mixture:
50 μl PCR assay was performed according to the following protocol.
Figure 1. DNA electrophoresis with amplified message
sequence (M1 and M2) 599 bp in size. C - is water control.
Conclusion:
Amplification of the message sequence 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 599 bp.
Procedure after gel validation:
PCR product was subsequently cleaned with PCR Purification Kit – (Jena Bioscience).
Restriction digestion
Experiment:
07/10/16: On this day restriction digestion of PCR product of the
message sequence and pSB1C3 (BBa_J04450) was done. Message
sequence as an insert was cut with EcoRI and PstI restriction enzymes.
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.
Figure 2. DNA electrophoresis with cut backbone pSB1C3
(BBa_J04450).
Conclusion:
RD of the PCR product of the message sequence and pSB1C3 (BBa_J04450)
was successful. RD of pSB1C3 (BBa_J04450) was verified by DNA
electrophoresis. Correct size of band could be seen and
that was 2019 bp.
07/10/16: Restriction digestion was followed by overnight
ligation. Vector (pSB1C3) and insert (message sequence) was ligated in
4:6 molar ratio.
Ligation mixture:
20 μl ligation assay was performed according to the following
protocol.
Transformation
Experiment:
08/10/16: On this day transformation into E. coli Top10 cells was done.
Selection was made on 50 μg/ml chloramphenicol LB agar plates.
Transformation protocol used can be found here.
09/10/16: 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 colony PCR was
performed (see colony PCR protocol). Primers message
prefix and message suffix were used for this colony PCR. Sequences of
these primers can be found here.
PCR mixture:
50 μl PCR assay was performed according to the following protocol.
Figure 3. DNA electrophoresis with samples from colony
PCR.Figure 4. Transformation of pSB1C3+message into E. coli
Top10.
Conclusion:
Transformation of pSB1C3+message into E. coli Top10 appeared to be
successful. Correct sizes of the bands from colony PCR were obtained: 599 bp. Correct clones were grown overnight (see cell culture protocol) from pre-glycerol stocks
made for colony PCR (see colony PCR protocol).
Validation
Experiment:
10/10/16: Grown cultures of E. coli Top10 with pSB1C3+message were taken
out from the incubator after overnight incubation. Glycerol stocks were made and plasmid isolation was performed (see
(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:
Plasmids pSB1C3+message from colonies 1, 2, 3, 4 and 5 were sent for sequencing with the sequencing
primers VF2 and VR (primer sequence can be found here).
Conclusion:
Sequencing results showed that cloning of message sequence into
pSB1C3 was successful. See sequencing results below.
Figure 5. Sequencing result of the message in
pSB1C3 with VF2 primer.Figure 6. Sequencing result of the message in
pSB1C3 with VF2 and VR primers.Figure 7. Sequencing result of the message in
pSB1C3 with VF2 and VR primers.Figure 8. Sequencing result 249 of the message in
pDR111.
Experiments
Experiments:
See integration of the message sequence into B. subtilis via the BioBrick BBa_K823023 integration plasmid in Proof of concept
experiment.
