The message sequence was ordered as a gBlock from IDT to construct the message 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 message sequence into the pDR111 in E. coli. pDR111 is an integration plasmid which can be integrated into the B. subtilis genome.
By double cross-over it replaces amyE gene, 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 message sequence into the B. subtilis 168 sub+. The other approach made use of the BioBrick integration plasmid BBa_K823023 (Message sequence in BBa_K823023).
PCR
Experiment:
25/07/16: The message sequence was amplified from gBlock ordered from IDT (Integrated DNA technologies).Primers used for the amplification were F-message sequence and R-message sequence (primer sequences can be found here).
PCR mixture:
50 μl PCR assay was performed according to the following
protocol.
Figure 2. DNA electrophoresis with amplified message sequence 572 bp in size.
Conclusion:
PCR of the message sequence from the IDT gBlock was successful. It was verified by DNA electrophoresis.
Correct size of a band could be seen and that was 572 bp.
Procedure after gel validation:
PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience
(see protocol).
Restriction digestion
Experiment:
26/07/16: On this day restriction digestion of PCR product of the message sequence and pDR111 integration plasmid was done. Message sequence as an insert was cut with SalI and HindIII restriction enzymes. Integration plasmid pDR111 as a vector was cut with exactly same restriction enzymes.
RD mixture:
20 μl RD assay was performed according to the following
protocol.
Figure 3. DNA electrophoresis with samples from restriction digestion.
Conclusion:
RD of the PCR product of message sequence and integration plasmid pDR111 was successful. It was verified by DNA electrophoresis. Correct size of bands could be seen and that was 7,834 bp for pDR111 and 572 bp for message sequence.
Procedure after gel validation:
Digested samples were cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin)
(see protocol).
Ligation
Experiment:
26/07/16: Restriction digestion was followed by overnight ligation. Vector (integration plasmid pDR111) and insert (message sequence) was ligated in 1:5 molar ratio.
Ligation mixture:
20 μl ligation assay was performed according to the following
protocol.
Transformation
Experiment:
27/07/16: This day transformation into E. coli Top10 and MC1061 cells was done. Correct clones were selected on 100 μg/ml ampicillin plates. Transformation protocol used can be found here.
28/07/16: Next day 12 colonies were obtained on the plates of E. coli MC1061 strain. To verify correct transformants colony PCR was performed (see Colony PCR protocol). Primers F-message sequence and R-message sequence were used for this colony PCR. Sequences of these primers can be found here.
PCR mixture:
25 μl PCR assay was performed according to the following
protocol.
PCR set-up:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
1:30 min
(30X)
72ºC
2:00 min
10ºC on hold
DNA electrophoresis:
For detailed information on how to prepare and run agarose gel see following protocol.
Figure 4. DNA electrophoresis with samples from colony PCR.Figure 5. Transformation of pDR111+message into E. coli MC1061.
Conclusion:
Transformation of pDR111+message into E. coli MC1061 appeared to be successful. Transformation efficiency was low, only 12 colonies were obtained, but 4 colonies seemed to be correct clones. And those were colonies 1, 8, 10 and 11 (see Figure 5). Correct sizes of the bands were obtained: 572 bp. Those colonies were grown overnight (see cell culture protocol) from pre-glycerol stocks made for colony PCR (see colony PCR protocol).
Validation
Experiment:
29/07/16: Grown cultures of E. coli MC1061 with pDR111+message were taken out from the incubator after overnight incubation. Glycerol stocks) were made and plasmid isolation was performed (see Mini prep protocol).
Firstly, concentration of the plasmids was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.
Sequencing:
Plasmids pDR111+message from colonies 1, 8, 10 and 11 were sent for sequencing)
with the sequencing primer Gb_insert_F2 ((see primer list)).
Conclusion:
Sequencing results showed that cloning of message sequence into integration plasmid pDR111 was successful. However just sequencing result 249 (Figure 9) has 100 % homology when compared to the reference (message sequence). Others have some bases missing or bases are not matching, it could be due to faulty sequencing. See sequencing results below.
Figure 6. Sequencing result 246 of the message in pDR111.Figure 7. Sequencing result 247 of the message in pDR111.Figure 8. Sequencing result 248 of the message in pDR111.Figure 9. Sequencing result 249 of the message in pDR111.
