Team:Groningen/PlasmidConstruction

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Acknowledgements

Plasmid construction

The following plasmids were constructed mainly during summer/autumn 2016.

Key sequence 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).

PCR

Experiment:

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.

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 the key PCR product sample 1-3. The correct size is 144 bp.
Conclusion:

PCR of the key sequence from the IDT gBlock was successful. We could see the correct band size which was 144 bp.

Procedure after gel validation:

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

Restriction digestion

Experiment:

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.

RD mixture:

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

DNA Electrophoresis:

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.

Figure 2. DNA electrophoresis of digested pDR111 with SalI, HindIII. The correct size is 7,834 bp.
Conclusion:

The restriction digestion was successful. We could see a band of 7,834 bp.

Procedure after gel validation:

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).

Ligation

Experiment:

31/08/16: The cut key was ligated to the SalI, HindIII cut pDR111.

Ligation mixture:

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

Transformation

Experiment:

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.

Figure 3. Transformation of the key sequence into the pDR111.
PCR mixture:

25 μl PCR assay was performed according to the following protocol.

PCR set-up:
95ºC2:00 min
95ºC30s(30X)
60ºC30s(30X)
72ºC30s(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. DNA electrophoresis of colony PCR for the key sequence in pDR111. Sample 5,6,7 and 9 show the correct size of 504 bp.
Conclusion:

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.

Validation

Experiment:

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.

Sequencing:

Plasmids pDR111+key from colonies 5, 6, 7 and 9 were sent for sequencing with the sequencing primer F message sequencing (see primer list).

Conclusion:

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.

Figure 5. Sequencing result of the key in pDR111 for sample 5, 6 and 9.

Experiments

Experiments:

See Proof of concept experiment.

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.

Key sequence in BBa_K823023

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 key strain. Construction was performed as described in the following.

PCR

Experiment:

27/09/16: The key sequence was amplified (see PCR protocol) from the pDR111+key plasmid with the primers key only + prefix and key only + suffix (primer sequences can be found here). The correct size of 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 sequence with prefix and suffix was successfully amplified from the gBlock. Band of 187 bp could be seen.

Procedure after gel validation:

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

Restriction digestion

Experiment:

04/10/16: The key sequence PCR product was digested with EcoRI and PstI as well as the BBa_K823023 plasmid. The digestion of the BBa_K823023 backbone was checked with DNA electrophoresis. The digestion of the key sequence PCR product was immediately cleaned with PCR Purification Kit – Jena Bioscience see protocol.

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. BBa_K823023 cut with EcoRI and PstI. Backbone ~6,000 bp, RFP insert ~1,000 bp.
Conclusion:

The digestion of BBa_K823023 showed the correct bands on the gel for the 6,000 bp backbone and the 1,000 bp RFP insert. Therefore the cloning procedure could proceed.

Procedure after gel validation:

Digested sample of the backbone ~6,000 bp were cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin) see protocol.

Ligation

Experiment:

04/10/16: The EcoRI and PstI cut BBa_K823023 integration backbone was ligated with the EcoRI, PstI cut key sequence.

Ligation mixture:

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

Transformation

Experiment:

04/10/16: 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 for the correct constructs. The next day colonies were selected to perform colony PCR in order to find the correct constructs.

Figure 3. Colonies of E.coli Top10 after transformation with BBa_K823023 with the key sequence.
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 gel see following protocol.

Conclusion:

The transformation of the key sequence in BBa_K823023 to E. coli Top10 was successful. We have obtained correct insert size when colony PCR was done. And that was 187 bp.

Validation

Experiment:

05/10/16 Grown cultures of E. coli Top10 with the key sequence in BBa_K823023 were used for making the (see Mini prep protocol). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Figure 4. Selecting colonies for glycerol stocks.

Experiments

Experiments:

See Proof of concept experiment.

Message sequence in pDR111

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.

PCR set-up:
95ºC2:00 min
95ºC30s(12X)
60ºC30s(12X)
72ºC1:30 min(12X)
72ºC2:00 min
10ºC on hold
DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see 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.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see 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:
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 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.

Experiments

Experiments:

See Proof of concept experiment.

Message sequence in pSB1C3 (BBa_K1930001)

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.

PCR set-up:
95ºC2:00 min
95ºC30s(30X)
60ºC30s(30X)
72ºC2:00 min(30X)
72ºC2:00 min
10ºC on hold
DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel 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. 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.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see 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.

Procedure after gel validation:

The upper band was cut out from the gel and DNA was extracted by Gel extraction kit (NucleoSpin® Gel and PCR Clean-up).

Ligation

Experiment:

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.

PCR set-up:
95ºC2:00 min
95ºC30s(30X)
60ºC30s(30X)
72ºC2:00 min(30X)
72ºC2:00 min
10ºC on hold
DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see 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.

Message sequence in BBa_K823023

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:
95ºC2:00 min
95ºC30s(30X)
60ºC30s(30X)
72ºC2:00 min(30X)
72ºC2:00 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.

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 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.

PCR set-up:
95ºC2:00 min
95ºC30s(30X)
60ºC30s(30X)
72ºC2:00 min(30X)
72ºC2:00 min
10ºC on hold
DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see 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.

Experiments

Experiments:

See Proof of concept experiment.

sfGFP(Sp) in pSB1C3 (BBa_K1930006)

sfGFP(Sp) is a reporter gene originally optimized for Streptococcus pneumoniae. It has been shown when expressed in B. subtilis that the signal is even brighter than the one from sfGFP(Bs) (Overkamp et al. 2013). To submit sfGFP(Sp) reporter gene as BioBrick (BBa_K1930006) we cloned it in the pSB1C3 standard iGEM backbone.

PCR

Experiment:

06/10/16: sfGFP(Sp) was amplified from the pDR111+sfGFP(Sp) plasmid, that we constructed during the project, with the primers prefix sfGFP(Sp) and suffix sfGFP(Sp) (primer sequences can be found here).

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 gel see following protocol.

Figure 1. DNA electrophoresis with amplified sfGFP(Sp) gene (763 bp in size).
Conclusion:

The sfGFP(Sp) was successfully amplified from the pDR111+sfGFP(Sp) plasmid.

Procedure after gel validation:

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

Restriction digestion

Experiment:

The sfGFP(Sp) 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:

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

DNA Electrophoresis:

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

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

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

Procedure after gel validation:

The upper band of 2000 bp was cut out from the gel and DNA was extracted with Agarose Gel Extraction Kit – Jena Bioscience. The PCR product was not checked on the gel after the digestion but immediately cleaned up with NucleoSpin® Gel and PCR Clean-up.

Ligation

Experiment:

The cut and cleaned sfGFP(Sp) was ligated to the cut and cleaned pSB1C3 in a ratio of 2:1.

Ligation mixture:

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

Transformation

Experiment:

07/10/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the transformation protocol. Cells were plated on 50 μg/ml chloramphenicol LB agar to select the correct construct.

09/10/16: Colonies were picked to perform colony PCR to find the correct constructs with the primers prefix sfGFP(Sp) and suffix sfGFP(Sp). Find primer sequences here.

Figure 3. Top10 E. coli colonies on 50 μg/ml chloramphenicol LB agar plates after transformation with sfGFP(Sp) 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 gel see following protocol.

Figure 4. DNA electrophoresis of the colony PCR product. C - is the water control. Samples 1-5 are from colony 1-5.
Conclusion:

All 5 samples show the right band by 763 bp. Therefore all of them were grown overnight to harvest the plasmid the following day.

Validation

Experiment:

10/10/16: Grown cultures of E. coli Top10 with the construct sfGFP(Sp) in pSB1C3 were used to obtain (glycerol stocks) and plasmid isolation was performed with QIAprep® Spin Miniprep Kit. Some of the overnight cultures seemed to already express the sfGFP (see Figure 5). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Figure 5. Pellet of the overnight cultures from sample 2 and 5. Looks like the sfGFP(Sp) is also expressed in E. coli Top10.
Sequencing:

Sequencing results showed that sfGFP(Sp) gene is present (see Figure 6). However something happened with the prefix and the suffix most probably during cloning steps. It looks that prefix and also suffix was disrupted by insertion of few base pairs. We cannot really explain what happened and due to time limitation we could not fix this problem. However sfGFP(Sp) is clearly expressed in E. coli (see Figure 5).

Figure 6. Sequencing result of sfGFP(Sp) in pSB1C3. GFP gene itself is underlined by red line.
Conclusion:

The sfGFP(Sp) was cloned to the backbone pSB1C3. However from the sequencing results we could see that this part contains bad prefix and suffix (see Figure 6). GFP gene is underlined by red line on Figure 6, however you could also see the bad prefix and suffix.

Experiments

See decoy experiments with sfGFP(Sp) in B. subtilis.

sfGFP(Sp) in pDR111 and in pDR111+message plasmid

sfGFP(Sp) is a reporter gene originally optimized for Streptococcus pneumoniae. It has been shown when expressed in B. subtilis that the signal is even brighter than the one from sfGFP(Bs) (Overkamp et al. 2013). sfGFP(Sp) was cloned either only to pDR111 integration plasmid or to pDR111+message plasmid. These plasmids were constructed with an interest to have easier screening when integrated into the B. subtilis genome. 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, with desired insert which is located between amyE front flanking region and amyE back flanking region. See the plasmid map below.

PCR

Experiment:

10/08/16: sfGFP(Sp) was amplified from pNW plasmid together with its promoter (Ppta), which is a constitutive promoter from Geobacillus DSM2542 for phosphate acetyl transferase expression, and a triple terminator (3TER). Primers used for the amplification were F-gfp insert and R-gfp insert (primer sequences can be found here).

PCR mixture:

25 μl PCR assay was performed according to the following protocol.

PCR set-up:
95ºC2:00 min
95ºC30s(30X)
57ºC30s(30X)
72ºC1:30 min(30X)
72ºC2:00 min
10ºC on hold
DNA Electrophoresis:

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

Figure 2. DNA electrophoresis with amplified Ppta-sfGFP(Sp)-3TER (1147 bp in size).
Conclusion:

Amplification of the Ppta-sfGFP(Sp)-3TER was successful. It was verified by DNA electrophoresis. Correct size of a band could be seen and that was 1147 bp. However second band could be seen, that was probably caused by unspecific binding of the primers used. Therefore lower band of correct size was cut out from the gel and DNA was extracted.

Procedure after gel validation:

DNA extraction was done according to this protocol.

Restriction digestion

Experiment:

10/08/16: On this day restriction digestion of PCR product of the sfGFP(Sp), pDR111 integration plasmid and pDR111+message integration plasmid was done. sfGFP(Sp) as an insert was cut with BglII and NheI restriction enzymes. Integration plasmid pDR111 and pDR111+message as a vector was cut with BamHI and NheI. BglII could not be used as a restriction enzyme for pDR111 due to multiple recognition site of this cutter. BamHI as a compatible restriction enzyme to BglII was used instead.

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 gel see following protocol.

Figure 3. DNA electrophoresis with samples from restriction digestion.
Figure 4. DNA electrophoresis with samples from restriction digestion.
Conclusion:

RD of the PCR product of sfGFP(Sp), integration plasmid pDR111 and pDR111+message was successful. RD of pDR111 and pDR111+message was verified by DNA electrophoresis. Correct sizes of bands could be seen and that was 6479 bp and 7034 bp, respectively.

Procedure after gel validation:

The upper band was cut out from the gel and DNA was extracted by Gel extraction kit (NucleoSpin® Gel and PCR Clean-up).

Ligation

Experiment:

10/08/16: Restriction digestion was followed by ligation at room temperature for 45 min. Vector (integration plasmid pDR111 and pDR111+message) and insert (sfGFP(Sp)) was ligated in 1:5 molar ratio.

Ligation mixture:

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

Transformation

Experiment:

10/08/16: Ligation was followed by transformation into E. coli Top10. Selection was made on 100 μg/ml ampicillin LB agar plates. Transformation protocol used can be found here.

11/08/16: 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 we grew some of the colonies overnight in 3 ml LB with 100 μg/ml ampicillin (see cell culture protocol). On the next day plasmid purification and restriction digestion control was done.

Conclusion:

Transformation of pDR111+sfGFP(Sp) and pDR111+message+sfGFP(Sp) into E. coli Top10 was assumed to be successful. To see if we obtained correct clones we did a restriction digestion control with EcoRI restriction enzyme.

Validation

Experiment:

12/08/16: Next day plasmid purification was done according to this protocol and restriction digestion control with EcoRI restriction enzyme was done to see if correct clones were obtained. This enzyme has 2 recognition sites in our construct. Therefore two bands (1535 bp and 6081 bp from pDR111+sfGFP(Sp) and 2090 bp and 6081 bp from pDR111+message+sfGFP(Sp)) should have been obtained on the agarose gel. Moreover we sent this new constructed plasmid for sequencing.

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 gel see following protocol.

Figure 5. DNA electrophoresis with samples (1-4 from pDR111+sfGFP(Sp)) from RD control with EcoRI.
Figure 6. DNA electrophoresis with samples (1-6 from pDR111++message+ sfGFP(Sp)) from RD control with EcoRI.
Sequencing:

Plasmid pDR111+sfGFP(Sp) and pDR111+message+sfGFP(Sp) was sent for sequencing. Primers used for the sequencing were F-gfp insert for pDR111+sfGFP(Sp) and F-gfp insert and F-message sequence for pDR111+message+sfGFP(Sp) (primer sequences can be found here).

Conclusion:

Figure 5 shows two correct bands (1535 bp and 6081 bp) when the pDR111+sfGFP(Sp) was cut. Figure 6 shows two correct bands (2090 bp and 6081 bp) when the pDR111+message+sfGFP(Sp) was cut. Therefore we assumed that our cloning was successful and correct plasmids were obtained. Sequencing results confirmed our assumption as you can see in Figure 7, 8 and 9.

Figure 7. Sequencing result 334 of the sfGFP(Sp) construct in pDR111 (primer used: F-gfp insert).
Figure 8. Sequencing result 335 of the sfGFP(Sp) construct in pDR111+message (primer used: F-gfp insert).
Figure 9. Sequencing result 336 of the sfGFP(Sp) construct in pDR111+message (primer used: F-message sequence).

Experiments

Experiment:

The functionality of these constructs was checked by integration into the B. subtilis genome 168 trp+ and imaging under the microscope.

Integration into the genome of B. subtilis 168 trp+:

sfGFP(Sp) in pDR111 and pDR111+message was integrated into the B. subtilis 168 trp+ genome. pDR111 is an integration plasmid which can be integrated into the B. subtilis genome. By double cross-over it replaces amyE gene, with desired insert, in this case insert is Ppta-sfGFP(Sp)-3TER, which is located between amyE front flanking region and amyE back flanking region.

Experiment set-up:

10 μl of sfGFP(Sp) in pDR111 and pDR111+message plasmids were transformed into the B. subtilis 168 trp+ strain (see transformation protocol). Selection was made on 150 μg/ml spectinomycin LB agar plates.

Microscopy experiment set-up:

B. subtilis cells were grown overnight in 3 ml LB with 150 μg/ml spectinomycin at 37°C and 220 rpm. On the next day microscopy slide was prepared as explained in the following protocol. Cells were imaged using phase contrast: filter POL 50% 1 s exposure, green fuorescence: FITC, 0.8 s exposure, objective: Olympus 100X/1.40, camera: CoolSNAP_HQ/HQ2-ICX285 and software: Resolve3D softWoRx-Acquire version.

Figure 10. B. subtilis 168 trp+ cells with sfGFP integrated into the amyE locus.
Conclusion:

Integration into the B. subtilis 168 trp+ strain of the plasmids with the reporter sfGFP(Sp) was successful. Multiple colonies were obtained on selection plates. B. subtilis cells were subsequently grown and checked under the microscope to see if there is an expression of GFP (see Figure 10). We could definitely see the GFP signal when observing the cells under the microscope. This was big enough proof that our plasmid construction followed by integration into the B. subtilis genome was successful.

PAtpI in pSB1C3 (BBa_K1930005)

The promoter PAtpI has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 min of the germination of B. subtilis (Sinai et al. 2015). atpA gene is part of an operon atpI-atpB-atpE-atpF-atpH-atpA-atpG-atpD-atpC, therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen. The region was checked for the binding of sigma factors and transcription factors with DBTBS. No binding factors were found with the highest significance level. In our project we wanted to find a constitutive promoter for our ciprofloxacin resistance casette. In the following part we put the promoter PAtpI in the pSB1C3 backbone to make it available to other iGEM teams.

PCR

Experiment:

06/10/16: The promoter PAtpI was amplified (see PCR protocol) from the ciprofloxacin resistance cassette BioBrick BBa_K1930004) with the primers pATPI+prefix and pATPI+suffix (primer sequences can be found here). The correct size of 372 bp for the PCR product was checked with 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 gel see following protocol.

Figure 1. DNA electrophoresis with amplified PAtpI promoter sequence 372 bp in size.
Conclusion:

The PAtpI promoter was successfully amplified with prefix and suffix from the plasmid.

Procedure after gel validation:

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

Restriction digestion

Experiment:

07/10/16: The PAtpI 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 protocol.

DNA Electrophoresis:

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

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

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

Procedure after gel validation:

The upper band of 2000 bp was cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin). The PCR product was not checked on the gel after the digestion but immediately cleaned up with (CR Purification Kit – Jena Bioscience.

Ligation

Experiment:

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

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 the correct constructs. The next day colonies were picked to perform colony PCR to find the correct constructs with the primers pATPI+prefix and pATPI+suffix (primer sequences can be found here).

Figure 3. E. coli Top10 transformed with PAtpI 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 gel see following protocol.

Figure 4. Result of the colony PCR. Samples from colony 1-5 are showing the correct size of the promoter PAtpI of 372 bp. C - is the water control.
Conclusion:

The transformation of PAtpI in pSB1C3 to E. coli Top10 was successful.

Validation

Experiment:

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

Sequencing:

The plasmid PAtpI in pSB1C3 from colonies 1 and 2 was sent for sequencing with the primers VF2 and VR, see primer list.

Figure 6. Sequencing result for PAtpI in pSB1C3 from colony 1 with VR.
Figure 5. Sequencing result for PAtpI in pSB1C3 from colony 1 with VF2.
Conclusion:

The sequencing result proofed the successful integration of the PAtpI promoter in the pSB1C3. Another BioBrick was obtained!

Experiments

For further experiments with this BioBrick see: MIC value experiment.

Ciprofloxacin resistance cassette in pSB1C3 (BBa_K1930004)

To design a qnrS1 resistance cassette BioBrick we designed a gBlock that contains the Bacillus subtilis constitutive promoter PAtpI, which is active from a very early stage of germination and includes a ribosome binding site. The gBlock also contains the original qnrS1 gene sequence from E. coli, the double terminator (BBa_B0015) from iGEM as well as the prefix and suffix for BioBricks. In summary the ciprofloxacin cassette consists of the following parts PAtpI+RBS+qnrS1+2TER, see plasmid map below.

PCR

Experiment:

The sequence of the qnrS1 gene was amplified from the gBlock qnrS1 E. coli ordered from IDT. Primers used for the amplification were F-qnrs1 E.coli and R-qnrs1 E.coli (primer sequences can be found here).

PCR mixture:

50 µl PCR assay was performed according to the following protocol.

PCR set-up:
95ºC2:00 min
95ºC30s(12X)
60ºC30s(12X)
72ºC1:30 min(12X)
72ºC2:00 min
10ºC on hold
DNA Electrophoresis:

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

Figure 1. Gel electrophoresis of the PCR product.
Conclusion:

The PCR of the qnrS1 sequence from the IDT gBlock was successful. It was verified by DNA electrophoresis. A band with the correct size of 1,194 bp could be seen.

Procedure after gel validation:

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

Restriction digestion

Experiment:

28/09/16 The qnrS1 gene should have been cloned into the pSB1C3. For this case the vector BBa_J04450 was used. The qnrS1 gene and BBa_J04450 were cut with the restriction enzymes EcoRI and PstI.

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.

Bands of the sizes 2,029 bp for the vector and 1,176 bp for the gene were expected.

Figure 2. DNA electrophoresis of EcoRI and PstI digested pSB1C3 - BBa_J04450. The RFP insert is 1,069 bp and the pSB1C3 backbone is 2019 bp.
Conclusion:

The digestion was successful because bands for both expected fragments could be seen on the gel, namely RFP insert is 1,069 bp and the pSB1C3 backbone is 2,019 bp.

Procedure after gel validation:

The digested samples were cut out from the gel and the DNA was extracted using the Agarose gel Extraction Kit (Jena Bioscience).

Ligation

Experiment:

28/09/16: After restriction digestion ligation was performed. 6 µl qnrS1 insert DNA were ligated to 4 µl pBS1C3 vector DNA. The ligation took place for 2 h at room temperature.

Ligation mixture:

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

Transformation

Experiment:

28/09/16 After ligation the ciprofloxacin resistance cassette in pSB1C3 was transformed into E. coli Top10 cells. The transformation was plated on plates containing 50 µg/ml chloramphenicol. The transformation was performed according to the transformation protocol.

29/09/16: To analyze the success of the transformation 12 samples were picked and a colony PCR was performed according to the following protocol. The primers F-qnrs1 E.coli and R-qnrs1 E.coli were used. Primer sequences can be found here.

DNA electrophoresis:

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

Conclusion:

The colony PCR did not bring clear results.

Experiment:

30/09/16: To further analyze the success of the transformation four colonies were grown as overnight cultures (started on 29/09/16) in LB and 50 µg/ml chloramphenicol. On the next day plasmid isolation was performed and plasmids were digested using the enzymes EcoRI and PstI according to the following restriction digestionprotocol.

DNA electrophoresis:

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

Conclusion:

Two out of the four digested samples showed bands of the expected sizes 2,029 bps for the vector and 1184 for the gene could be seen.

Validation

Experiment:

The two samples that showed the correct digestion pattern in the restriction digestion control were sent for sequencing with the primers VF2 and VR, see primer list. These two samples showed the correct sequence (Figure 5.1-5.4, 5.1 mutant 1 forward, 5.2: mutant 2 forward, 5.3: mutant 1 reverse, 5.4: mutant 2 reverse ).

Figure 5.1
Figure 5.2
Figure 5.3
Figure 5.4

Experiments

For further experiment see the construction of the plasmid ciprofloxacin resistance cassette in BBa_K823023.

Ciprofloxacin resistance cassette in BBa_K823023

To design a ciprofloxacin resistance cassette we designed a gBlock that contains the Bacillus subtilis promoter PAtpI, which is active from a very early stage of germination and includes a ribosome binding site. The gBlock also contains the original qnrS1 gene sequence from E. coli. qnr genes code for pentapeptide repeat proteins. These proteins reduce susceptibility to quinolones by protecting the complex of DNA and DNA gyrase enzyme from the inhibitory effect of quinolones. Finally, this gBlock contains a double terminator BBa_B0015 from iGEM as well as the prefix and suffix for BioBricks. In summary the ciprofloxacin cassette consists of the following parts PAtpI+RBS+qnrS1+2TER.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. Construction of ciprofloxacin resistance cassette in BBa_K823023 integration plasmid was performed as described in the following.

PCR

Experiment:

The sequence of the qnrS1 gene was amplified from the gBlock qnrS1 E. coli ordered from IDT. Primers used for the amplification were F-qnrs1 E.coli and R-qnrs1 E.coli/ (primer sequences can be found here).

PCR mixture:

50 µl PCR assay was performed according to the following protocol.

PCR set-up:
95ºC2:00 min
95ºC30s(12X)
60ºC30s(12X)
72ºC1:30 min(12X)
72ºC2:00 min
10ºC on hold
DNA Electrophoresis:

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

Figure 1. DNA electrophoresis of the PCR product.
Conclusion:

The PCR of the qnrS1 sequence from the IDT gBlock was successful. It was verified by DNA electrophoresis. A band with the correct size of 1,194 bp could be seen.

Procedure after gel validation:

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

Restriction digestion

Experiment:

28/09/16: The qnrS1 PCR product and BBa_K823023 were cut with the restriction enzymes EcoRI and PstI.

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. BBa_K823023 cut with EcoRI and PstI. Backbone ~6,000 bp, RFP insert 1000 bp.
Conclusion:

The digestion of BBa_K823023 showed the correct bands on the gel. 6,000 bp backbone for the backbone and the 1000 bp for the RFP insert. Therefore the cloning procedure could proceed.

Procedure after gel validation:

Digested sample of the backbone ~6,000 bp were cut out from the gel and DNA was extracted by Agarose gel extraction kit (Jena Bioscience) (see protocol).

The digestion of the qnrS1 PCR product was immediately cleaned with the kit (PCR Purification Kit – Jena Bioscience).

Ligation

19/10 In this ligation the EcoRI, PstI cut gene qnrS1 and the vector BBa_K823023 were combined.

Ligation mixture:

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

Transformation

Experiment:

29/09/16: 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 for the correct constructs. On the next day colonies were selected to perform colony PCR in order to find the correct constructs using the primers F-qnrs1 E.coli and R-qnrs1 E.coli. Primer sequences can be found here.

Figure 3. Transformation to E. coli Top10.
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 gel see following protocol.

Figure 4. Result of the colony PCR. Sample indicated with the star shows the correct sie of 1194 bp. C - is the water control.
Conclusion:

The transformation of qrnS1 (ciprofloxacin resistance cassette) in BBa_K823023 to E. coli Top10 was successful.

Validation

Experiment:

To test if the construct would make B. subtilis resistant to ciprofloxacin, the construct qrnS1 in BBa_K823023 was transformed into the B. subtilis 168 tpr+.

Experiments

Experiment:

13/10/16: The transformation to B. subtilis was performed according to the following protocol. Colonies were selected on LB agar with 5 μg/ml chloramphenicol.

Figure 5. B. subtilis after transformation with ciprofloxacin resistance cassette.

14/10/16: Colonies were streaked out on agar with starch to perform the starch test, which verifies the integration in the amyE locus in the genome of B. subtilis. Integration check: Starch test

Figure 6. Starch test. Colonies without a clear halo are positive for integration.
Conclusion:

The integration of the ciprofloxacin resistance cassette was successful.

As a first check on the functionality of the ciprofloxacin resistance cassette, we grew B. subtilis colonies from the starch test with ciprofloxacin. As a control they were also grown with chloramphenicol, the resistance on the backbone of the integration vector. Figure 7 shows the result for 3 different colonies (tubes indicated with 1 - 3). The tubes marked with Cm is the control with chloramphenicol, which shows growth for all three colonies. The tubes marked with Cipro were grown with ciprofloxacin. Colonies 2 and 3 showed growth, whereas colony 1 did not grow. Seems like the resistance cassette is working. To further explore if the ciprofloxacin cassette is functional in B. subtilis, a MIC value test was performed. See link.

Figure 7.

RBS+nucA in pSB1C3

One approach to delete the key from the genome of B. subtilis is making use of the nucA BioBrick (BBa_K729004). The nucA is a nuclease gene which has its origin in the genome of Staphylococcus aureus. It is capable of digesting genetic material. The RBS is from BioBrick (BBa_B0030). The combination of these two BioBricks is the first step of achieving our nucA killswitch, which consist of the tetR repressible promoter (BBa_R0040), the RBS controlling the nucA gene (BBa_B0030) and double terminator (BBa_B0015).

Obtaining RBS (BBa_B0030) and nucA (BBa_K729004)

Experiment:

01/08/16: The RBS (BBa_B0030) was obtained from the part distribution 2016, the plasmid was transformed to E. coli Top10 using this protocol. The nucA (BBa_K729004) was requested from the iGEM headquarters. Colonies from these transformations were cultured in LB medium. Grown cultures of E. coli Top10 with RBS and nucA were used to obtain a glycerol stocks and plasmid isolation was performed (QIAprep® Spin Miniprep Kit). The concentration of the plasmids obtained was measured on Nanodrop. The plasmids were stored at -20°C.

Restriction digestion

Experiment:

28/09/16: On this day restriction digestion of RBS (BBa_B0030) and nucA (BBa_K729004) was performed. The backbone RBS (BBa_B0030) was digested with SpeI and PstI. The insert nucA was digested with XbaI and PstI.

RD mixture:

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

DNA Electrophoresis:

The digestion mixture of backbone RBS in pSB1C3 and insert nucA was loaded on a gel to extract both parts. For detailed information on how to prepare and run agarose gel see following protocol.

Figure1 DNA electrophoresis of: 1. nucA in pSB1C3 cut with XbaI and PstI. The fragments are supposed to be nucA/ 561 bp and pSB1C3 2,019 bp. 2. RBS in pSB1C3 cut with SpeI and PstI one fragment of 2,070 bp
Conclusion:

The digestion was successful because the band of the nucA could be seen: 500 bp and RBS in pSB1C3 showed a band of 2,000 bp.

Procedure after gel validation:

Digested nucA and RBS in pSB1C3 were cut out from the gel and DNA was extracted by (Agarose Gel Extraction Kit – Jena Bioscience).

Ligation

Experiment:

28/09/16: The cut nucA was ligated to the SpeI and PstI cut RBS in pSB1C3.

Ligation mixture:

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

Transformation

Experiment:

28/09/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the transformation protocol. Cells were plated on 50 μg/ml chloramphenicol LB agar to select the correct constructs. The next day colonies were picked to perform colony PCR to find the correct constructs with the primers VF2 and VR. Find primers here.

Figure 2. Transformation of nucA in RBS 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)
72ºC30s(30X)
72ºC2:00 min
10ºC on hold
DNA electrophoresis:

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

Figure 3. DNA electrophoresis of colony PCR for samples 1-7 of RBS+nucA in pSB1C3 with primers VF2, VR. Expected product 896 bp.
Conclusion:

Transformation of RBS+nucA in pSB1C3 in E. coli Top10 appeared to be successful. The samples 2, 5, 6 and 7 showed the right size of band. These samples were used to obtain the plasmid from an overnight culture.

Validation

Experiment:

29/09/16: Grown cultures of E. coli Top10 with RBS+nucA in pSB1C3 were used to obtain the 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.

Sequencing:

Plasmids RBS+nucA in pSB1C3 from colonies 2, 5, and 7 were sent for sequencing with the sequencing primers VF2 and VR. (See primer list).

Conclusion:

Sequencing results showed that the nucA was cloned in the pSB1C3 backbone but the RBS was missing and the suffix was also incorrect. See Figure 4. The nucA (BBa_K729004) received from the iGEM headquarters was sent for sequencing as well. It turned out that the part BBa_K729004 does have the nucA gene but the prefix and suffix are incorrect . See sequencing results in Figure 5 to 7.

Figure 4. Sequencing result of the RBS+nucA in pSB1C3 for sample 5, 7 and 9. nucA can be seen in pink. The prefix is marked in red.
Figure 5. Sequencing result of the BBa_K729004 aligned with the nucA gene.
Figure 6. Sequencing result of the BBa_K729004 aligned with the prefix.
Figure 7. Sequencing result of the BBa_K729004 aligned with the suffix.

Experiments

We wanted to improve the BBa_K729004 part by giving it the correct prefix and suffix. Unfortunately we did not succeed to complete this task.

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