Template:Groningen/Labjournal/Key-in-pDR111
The key sequence was ordered as a gBlock from IDT to construct the key B.
subtilis strain. In a first approach this was done with the pDR111 B.
subtilis integration plasmid, which can also be amplified by E. coli.
Therefore the first step was to clone the key sequence into the pDR111 in E. coli. pDR111 is an integration plasmid which can be integrated into the B. subtilis genome.
It replaces amyE gene by double cross-over, which is necessary for production of alpha-amylase
(see Subtiwiki.uni-goettingen.de) with desired insert which is located between amyE front flanking region and amyE back flanking region. See the plasmid map below. We used this plasmid for integration of our key sequence into the B. subtilis 168 sub+. The other approach made use of the BioBrick integration plasmid BBa_K823023 (Key sequence in BBa_K823023). 31/08/16: Amplification of the key sequence from the gBlock from IDT (see gBlocks protocol)
with the primers F key only amplify and R key only amplify (see primer list) was done. The correct size of the key PCR product of 144 bp was checked with DNA electrophoresis. 50 μl PCR assay was performed according to the following
protocol. For detailed information on how to prepare and run agarose gels see
following protocol. PCR of the key sequence from the IDT gBlock was successful. We could see the correct band size which was 144 bp. PCR product was subsequently cleaned with the kit (PCR Purification Kit – Jena Bioscience). 31/08/16: On this day restriction digestion of the PCR product of the key
sequence and pDR111 integration plasmid was performed. The key sequence
as an insert was cut with SalI and HindIII restriction enzymes. The
integration plasmid pDR111 as a vector was cut with exactly same
restriction enzymes. 20 μl RD assay was performed according to the following see
following protocol. The digestion mixture of backbone pDR111 was loaded on a gel to
extract the backbone. For detailed information on how to prepare and run
agarose gels see following protocol. The restriction digestion was successful. We could see a band of 7,834 bp. Digested pDR111 was cut out from the gel and DNA was extracted by Gel
extraction kit (Agarose Gel Extraction Kit – Jena Bioscience). The PCR product of the key was not
checked on the gel after the digestion but immediately cleaned up with
DNA Clean-up (NucleoSpin® Gel and PCR Clean-up). 31/08/16: The cut key was ligated to the SalI, HindIII cut pDR111. 20 μl ligation assay was performed according to the following protocol. 31/08/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the protocol.
Cells were plated on 100 μg/ml ampicillin LB agar to select the correct
construct. The next day colonies were picked to perform colony PCR to
find the correct construct with the primers F-amyE and R-amyE. Find
primers here. 25 μl PCR assay was performed according to the following
protocol. For detailed information on how to prepare and run agarose gels see
the following protocol. Transformation of pDR111+key into E. coli Top10 appeared to be
successful. The samples 5, 6, 7 and 9 showed the right size of band in
the colony PCR. These samples were used to obtain the plasmid from an overnight culture. 02/09/16: Grown cultures of E. coli Top10 with pDR111+key were used to
obtain a glycerol stocks and plasmid isolation was performed
(QIAprep® Spin Miniprep Kit). Firstly, concentration of the plasmids obtained was
measured on Nanodrop. Secondly, plasmids were sent for sequencing and
then stored at -20°C. Plasmids pDR111+key from colonies 5, 6, 7 and 9 were sent for
sequencing with the sequencing primer F message
sequencing (see primer list). Sequencing results showed that cloning of message sequence into the
integration plasmid pDR111 was successful. Sample 5, 6 and 9 show a
100% homology, so the key was successfully cloned into the pDR111
plasmid. See sequencing results in Figure 5. Key sequence in pDR111
PCR
Experiment:
PCR mixture:
DNA Electrophoresis:
Conclusion:
Procedure after gel validation:
Restriction digestion
Experiment:
RD mixture:
DNA Electrophoresis:
Conclusion:
Procedure after gel validation:
Ligation
Experiment:
Ligation mixture:
Transformation
Experiment:
PCR mixture:
PCR set-up:
95ºC 2:00 min 95ºC 30s (30X) 60ºC 30s (30X) 72ºC 30s (30X) 72ºC 2:00 min 10ºC on hold DNA Electrophoresis:
Conclusion:
Validation
Experiment:
Sequencing:
Conclusion:
