BBa_K823023 is an available BioBrick from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for
cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more
efficient screening after transformation. It was chosen to
construct the Bacillus subtilis message strain. Construction was
performed as described in the following.
PCR
Experiment:
06/10/16: 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.
PCR set-up:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
2:00 min
(30X)
72ºC
2:00 min
10ºC on hold
DNA Electrophoresis:
For detailed information on how to prepare and run agarose gels see
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 (see above). Correct size of a band could be seen and
that was 599 bp.
07/10/16: On this day restriction digestion of PCR product of the
message sequence and BBa_K823023 was done. Message sequence
as an insert was cut with EcoRI and PstI restriction enzymes.
BBa_K823023 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 gels see following protocol.
Figure 2. DNA electrophoresis with cut backbone
BBa_K823023.
Conclusion:
RD of the PCR product of message sequence and BBa_K823023 was
successful. RD of BBa_K823023 was verified by DNA electrophoresis. Correct size of band could be seen and that was ∼6000 bp.
Procedure after gel validation:
The upper band was cut out from the gel and DNA was extracted by Agarose gel
extraction kit (Jena Bioscience).
Ligation
Experiment:
07/10/16 Restriction digestion was followed by overnight
ligation. Vector (BBa_K823023) 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 was done.
Selection was made on 100 μg/ml ampicillin 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 BBa_K823023+message into
E. coli Top10.
Conclusion:
Transformation of BBa_K823023+message into E. coli Top10 appeared to
be successful. Correct sizes of the bands from colony PCR (see Figure
3) 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 BBa_K823023+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) protocol). Concentration of the
plasmids obtained was measured on Nanodrop and plasmids were stored at
-20°C.
