Experiments
Intro
Intro
We chose to save and transmit our encrypted message and the associated key in the genome of B. subtilis, which is our CryptoGErM. The message and key were generated by our Encryption machine. For the integration we tried two different integration plasmids, one is the BioBrick BBa_K823023 from iGEM Munich 2012 and the other one is the pDR111. Construction of the plasmids can be seen here (link to plasmid construction). Both plasmids integrate in the amyE locus of B. subtilis. The amyE gene, which codes for a starch degrading enzyme, is destroyed in the process of integration and therefore colonies can be screened with the starch test for successful integration. Both plasmids were constructed for both message and key however the following part will only focus on the BBa_K823023 integration plasmid.
The transformation into the B. subtilis was performed according to the protocol Transformation of B. subtilis with the key sequence in BBa_K823023 and message sequence in BBa_K823023. Since B. subtilis is naturally competent it is easy and efficient to integrate the message/key into the genome of this bacterium. We also tested the transformation efficiency of the BBa_K823023 backbone (link to transformation efficiency K823023).
The obtained colonies were screened for successful integration with the starch test Integration check: Starch test.
The starch test in Figure 2 shows that almost all colonies are not able to degrade starch therefore it can be assumed that they integrated our message/ key into the amyE locus. The starch test offers a quick and cheap way to screen multiple colonies.
Now, that we had a B. subtilis strain with our message and another one with our key, we performed the sporulation protocol Preparation of the spore stock of B. subtilis to obtain a spore batch carrying the key and another one with the message.
At this point it was time to find out if we could actually send our message spores somewhere and retrieve the message back. The same procedure works for the key.
29/08. First the sending of the spores was simulated in the lab by leaving the 10 μl of message spores in a tube in an envelope for 24 h.
30/08. After the sending simulation, the spores were streaked out on LB agar with 150 μg/ml spectinomycin LB agar plates.
01/09. A single colony was picked from the restreaked spores plate and used as template for colony PCR to amplify the message sequence. Colony PCR with the F message sequence, R message sequence primers and F message sequencing, R message sequencing primers (primer sequences can be found in our primer list).
The expected size for the message product of the first primer pair was 572 bp and for the second one it is 916 bp. Both bands can be seen on the agarose gel (see Figure 5). The key product was 178 bp and the gel shows that this product was successfully amplified from the genome of B. subtilis. The PCR product was subsequently cleaned up with the kit DNA Clean-up (PCR Purification Kit – Jena Bioscience). The sample 3n and 4n were sent for sequencing with the primers F/R message sequencing Sequencing (Macrogen). Sequencing (Macrogen).
05/09. The moment of truth! We copied-pasted the encrypted message sequence and the key sequence in our decryption machine. The key is converted to plain text. For this proof of conept we chose the key “Autoclave after reading”. The result of the decryption was: The world is full of obvious things which nobody by any chance ever observes.” CryptoGErM works! We successfully integrated an encrypted message into the genome of B. subtilis, the key in another strain and received the same message back from the spores. As instructed by the key, all cryptoGErMs were autoclaved in the end of the experiments. .
The next step was to actually send the message to another iGEM team to demonstrate the functionality under real world conditions. Therefore read Collaborations.
The spores containing the DNA sequence that encodes our key will be sent in a mixture with decoy spores. These strains were constructed with our BioBricks BBa_K1930002 (key) and BBa_K1930006 (sfGFP) The high ratio of decoy spores makes it hard for unauthorized parties to retrieve the correct key if they try to sequence the entire sample by brute force. In this experiment we tried to determine how fine our system is in selecting the spores from the decoy once the right treatment is applied.
We prepared a Bacillus subtilis strain containing a superfolder GFP and a spectinomycin resistance cassette in the genome. Then we prepared mixtures of that mutant with wild-type B. subtilis in different ratios. In this experiment living cells were used instead of spores. After growing in different conditions, the final mixture of mutant vs wild-type strains was determined microscopically and in a flow cytometer.
Due to the high ratio of decoy cells, any non-approved party trying to sequence the entire sample will not be able to distinguish the key-sequence from the background noise. In the scientific literature, using standard sequencing techniques it has been possible to detect one mutant out of 150 wild-type molecules [1][2]. However, fine-tuned technologies, such as Duplex Sequencing, have shown to increase that number to one mutant in 10,000 wild-type cells. That same technique is theoretically able to detect one mutant out of 10 million decoys[3]!
We combined different ratios of sfGFP bacteria and decoy bacteria. LB medium was inoculated from glycerol stocks of the sfGFP strain and the wild-type strain which were grown overnight at 37 °C, shaking at 220 rpm in a 3 ml culture. On the next day the corresponding dilutions were made and grown again overnight at 37 °C in a shaking liquid 3 ml culture. The antibiotic was added to both the preculture and the diluted culture.
The next morning the cells were visualized in the microscope Time-lapse microscopy/Phase-contrast microscopy and additionally diluted 50 times in 1X PBS buffer to analyze in the flow cytometer.
Figure no. | Concentration spectinomycin [µg/ml] | Initial ratio sfGFP:decoy | Final ratio sfGFP:decoy |
---|---|---|---|
1,2 | 0 | 0:1 | 0:1 |
3,4 | 0 | 1:0 | 25:1 |
5,6 | 150 | 0:1 | 0:1 |
7,8 | 150 | 1:0 | 160:1 |
9,10 | 0 | 1:1 | 12:1 |
11,12 | 150 | 1:1 | 230:1 |
13,14 | 0 | 1:150 | 10:1 |
15,16 | 150 | 1:150 | 70:1 |
17,18 | 0 | 1:10,000,000 | 0:1 |
19,20 | 150 | 1:10,000,000 | 0:1 |
The mutant strain containing the superfolder GFP can be seen green in the microscopy images and is also marked green in the flow cytometer graphs. The wild-type decoy cells are gray in both cases.
As control groups we used samples that contained either only the decoy wild-type strain or the spectinomycin-resistant sfGFP mutant. While the wild type grew well without addition of spectinomycin (Figures 1 and 2), no growth could be observed if the antibiotic was added (Figures 5 and 6). On the other hand, the spectinomycin resistant sfGFP strain grew well both in its presence or absence (Figures 3, 4, 7 and 8). In both cases, growth of cells not expressing sfGFP was observed. This could be due to not fully developed cells that do not yet express sfGFP in their current cell cycle.
A mixed culture in the ratio 1:1 without the addition of spectinomycin showed presence of both wild-type and sfGFP cells (Figures 9, 10, 11 and 12) as expected. However, the unexpected higher ratio of sfGFP strain under conditions that do not give advantage over the wild-type strain leads us to assume that the mutant generally grows faster than the wild-type strain. Similarly, adding the antibiotic increases 20 times the fraction of sfGFP cells, in this case by killing the non-resistant wild-type.
The samples with a 1:150 ratio showed consistent results (Figures 13, 14, 15 and 16) compared to the 1:1 ratios. Without the addition of spectinomycin the mutant outgrew the wild-type strain (10:1) even though the initial ratio was not in its favor (1:150).
We went to an extreme of using a ratio of 1 mutant in 10 million wild-type cells. In this conditions, no growth of mutants was observed. The ratio is too high to allow the mutant strain to grow even in the presence of antibiotic that would give it an advantage over the wild-type strain.
Our experiment shows that a specific strain, in this case containing a sfGFP and a spectinomycin resistance cassette can be selected from a larger number of decoys. We could not determine the optimal ratio that would strengthen this layer of biosecurity. For further experiments the ratio of decoys should be fine tuned to determine the maximum ratio of spores:decoys that could be used, thus reassuring that unauthorized parties will not be able to recover the key by sequencing the whole sample but the intended recipient will still be able to recover it.
Here you can find how we constructed our BioBricks and other plasmids which were used during our project, next you can find protocols which we followed during our experiments and finally you can see our primer list with every primer used.
The following plasmids were constructed mainly during summer/autumn 2016.
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.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 30s | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
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.
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.
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.
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.
The key was successfully amplified with prefix and suffix from the pDR111+key plasmid.
PCR product was subsequently cleaned with (PCR Purification Kit – Jena Bioscience).
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.
30 μl RD assay was performed according to the following see following protocol.
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.
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.
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.
07/10/16: The EcoRI, PstI cut pSB1C3 backbone was ligated with the EcoRI, PstI cut key.
20 μl ligation assay was performed according to the following protocol.
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.
25 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 1:30 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gels see the following protocol.
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.
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.
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).
The sequencing result proofed the successful integration of the key into the pSB1C3. First BioBrick was made!
See integration of the key sequence in B. subtilis via the BioBrick BBa_K823023 integration plasmid in Proof of concept experiment.
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.
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.
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.
The key sequence with prefix and suffix was successfully amplified from the gBlock. Band of 187 bp could be seen.
PCR product was subsequently cleaned with (PCR Purification Kit – Jena Bioscience).
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.
20 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gels see following protocol.
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.
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.
04/10/16: The EcoRI and PstI cut BBa_K823023 integration backbone was ligated with the EcoRI, PstI cut key sequence.
20 µl ligation assay was performed according to the following protocol.
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.
25 µl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 1:30 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
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.
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.
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).
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).
50 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (12X) |
60ºC | 30s | (12X) |
72ºC | 1:30 min | (12X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
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.
PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience (see protocol).
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.
20 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gel see following protocol.
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.
Digested samples were cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin) (see protocol).
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.
20 μl ligation assay was performed according to the following protocol.
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.
25 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 1:30 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
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).
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.
Plasmids pDR111+message from colonies 1, 8, 10 and 11 were sent for sequencing) with the sequencing primer Gb_insert_F2 ((see primer list)).
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.
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.
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).
50 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 2:00 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
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.
PCR product was subsequently cleaned with PCR Purification Kit – (Jena Bioscience).
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.
20 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gel see following protocol.
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.
The upper band was cut out from the gel and DNA was extracted by Gel extraction kit (NucleoSpin® Gel and PCR Clean-up).
07/10/16: Restriction digestion was followed by overnight ligation. Vector (pSB1C3) and insert (message sequence) was ligated in 4:6 molar ratio.
20 μl ligation assay was performed according to the following protocol.
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.
50 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 2:00 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
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).
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.
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).
Sequencing results showed that cloning of message sequence into pSB1C3 was successful. See sequencing results below.
See integration of the message sequence into B. subtilis via the BioBrick BBa_K823023 integration plasmid in Proof of concept experiment.
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.
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).
50 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 2:00 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gels see following protocol.
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.
PCR product was subsequently cleaned with (PCR Purification Kit – Jena Bioscience).
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.
20 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gels see following protocol.
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.
The upper band was cut out from the gel and DNA was extracted by Agarose gel extraction kit (Jena Bioscience).
07/10/16 Restriction digestion was followed by overnight ligation. Vector (BBa_K823023) and insert (message sequence) was ligated in 4:6 molar ratio.
20 μl ligation assay was performed according to the following protocol.
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.
50 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 2:00 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
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.
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.
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.
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).
50 μl PCR assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gel see following protocol.
The sfGFP(Sp) was successfully amplified from the pDR111+sfGFP(Sp) plasmid.
PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience.
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.
20 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gel see following protocol.
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.
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.
The cut and cleaned sfGFP(Sp) was ligated to the cut and cleaned pSB1C3 in a ratio of 2:1.
20 μl ligation assay was performed according to the following protocol.
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.
25 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 1:30 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
All 5 samples show the right band by 763 bp. Therefore all of them were grown overnight to harvest the plasmid the following day.
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.
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).
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.
See decoy experiments with sfGFP(Sp) in B. subtilis.
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.
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).
25 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
57ºC | 30s | (30X) |
72ºC | 1:30 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
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.
DNA extraction was done according to this protocol.
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.
20 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gel see following protocol.
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.
The upper band was cut out from the gel and DNA was extracted by Gel extraction kit (NucleoSpin® Gel and PCR Clean-up).
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.
20 μl ligation assay was performed according to the following protocol.
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.
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.
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.
20 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gel see following protocol.
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).
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.
The functionality of these constructs was checked by integration into the B. subtilis genome 168 trp+ and imaging under the microscope.
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.
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.
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.
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.
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.
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.
50 μl PCR assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gel see following protocol.
The PAtpI promoter was successfully amplified with prefix and suffix from the plasmid.
PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience.
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.
30 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gel see following protocol.
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.
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.
07/10/16: The EcoRI, PstI cut pSB1C3 backbone was ligated with the EcoRI, PstI cut promoter PAtpI.
20 μl ligation assay was performed according to the following protocol.
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).
25 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 1:30 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
The transformation of PAtpI in pSB1C3 to E. coli Top10 was successful.
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.
The plasmid PAtpI in pSB1C3 from colonies 1 and 2 was sent for sequencing with the primers VF2 and VR, see primer list.
The sequencing result proofed the successful integration of the PAtpI promoter in the pSB1C3. Another BioBrick was obtained!
For further experiments with this BioBrick see: MIC value experiment.
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.
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).
50 µl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (12X) |
60ºC | 30s | (12X) |
72ºC | 1:30 min | (12X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gels see following protocol.
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.
PCR product was subsequently cleaned with (PCR Purification Kit – Jena Bioscience).
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.
20 μl RD assay was performed according to the following protocol.
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.
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.
The digested samples were cut out from the gel and the DNA was extracted using the Agarose gel Extraction Kit (Jena Bioscience).
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.
20 µl ligation assay was performed according to the following protocol.
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.
For detailed information on how to prepare and run agarose gel see following protocol.
The colony PCR did not bring clear results.
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.
For detailed information on how to prepare and run agarose gel see following protocol.
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.
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 ).
For further experiment see the construction of the plasmid 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.
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).
50 µl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (12X) |
60ºC | 30s | (12X) |
72ºC | 1:30 min | (12X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gels see following protocol.
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.
PCR product was subsequently cleaned with (PCR Purification Kit – Jena Bioscience).
28/09/16: The qnrS1 PCR product and BBa_K823023 were cut with the restriction enzymes EcoRI and PstI.
20 μl RD assay was performed according to the following protocol.
For detailed information on how to prepare and run agarose gels see following protocol.
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.
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).
19/10 In this ligation the EcoRI, PstI cut gene qnrS1 and the vector BBa_K823023 were combined.
20 µl ligation assay was performed according to the following protocol.
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.
25 µl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 1:30 min | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see following protocol.
The transformation of qrnS1 (ciprofloxacin resistance cassette) in BBa_K823023 to E. coli Top10 was successful.
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+.
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.
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
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.
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).
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.
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.
20 μl RD assay was performed according to the following protocol.
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.
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.
Digested nucA and RBS in pSB1C3 were cut out from the gel and DNA was extracted by (Agarose Gel Extraction Kit – Jena Bioscience).
28/09/16: The cut nucA was ligated to the SpeI and PstI cut RBS in pSB1C3.
20 μl ligation assay was performed according to the following protocol.
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.
25 μl PCR assay was performed according to the following protocol.
95ºC | 2:00 min | |
95ºC | 30s | (30X) |
60ºC | 30s | (30X) |
72ºC | 30s | (30X) |
72ºC | 2:00 min | |
10ºC on hold |
For detailed information on how to prepare and run agarose gel see the following protocol.
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.
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.
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).
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.
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.
All protocols which were used throughout our project can be found below or downloaded here.
Completed 1X MC Medium | |
---|---|
H2O | 1.8 ml |
10X MC Medium | 200 µl |
MgSO4 | 6.7 µl |
1 % tryptophan (for trp- strains) | 10 µl |
10X MC medium(for 100 ml) | |
---|---|
K2HPO4.3H2O | 14 g |
KH2PO4 | 5.2 g |
Glucose | 20 g |
300 mM Tri-Na citrate* | 10 ml |
Ferric NH4 citrate** | 1 ml |
Casein hydrolysate | 1 g |
K glutamate | 1 g |
*300 mM Tri-Na citrate = 8.8 g in 100 ml H2O (wrap in aluminium foil)
**Ferric NH4 citrate = 2.2 g in 100 ml H2O (wrap in aluminium foil)
Reagents supplied:
5X TBE buffer (for 1000 ml) | |
---|---|
Tris-base | 54 g |
Boric acid | 27.5 g |
0.5 M EDTA (pH 8.0) | 20 ml |
50 µl PCR assay | |
---|---|
10X Taq Reaction buffer complete | 5 µl |
dNTP Mix | 1 µl |
MgCl2* | 1 µl |
10 µM Forward Primer | 1 µl |
10 µM Reverse Primer | 1 µl |
Template DNA | 2 - 50 ng |
Taq polymerase | 0.2 – 0.5 µl |
MiliQ water | up to 50 µl |
*Adding of Mg2+ is recommended for most applications.
Cycling conditions | |||
---|---|---|---|
initial denaturation | 94°C | 2 min | 1x |
denaturation | 94°C | 30 sec | 30x |
annealing* | 45 – 68°C | 30 sec | 30x |
elongation** | 72°C | 30 sec – 4 min | 30x |
final elongation | 72°C | 2 min | 1x |
hold | 4 – 10°C |
*The annealing temperature depends on the melting temperature of the primers used.
**The elongation time depends on the length of the fragments to be amplified (1 min/kb).
50 µl PCR assay | |
---|---|
Q5 High-Fidelity 2X Master Mix | 25 µl |
10 µM Forward Primer | 2.5 µl |
10 µM Reverse Primer | 2.5 µl |
Template DNA | variable |
MiliQ water | up to 50 µl |
25 µl PCR assay | |
---|---|
Q5 High-Fidelity 2X Master Mix | 12.5 µl |
10 µM Forward Primer | 1.25 µl |
10 µM Reverse Primer | 1.25 µl |
Template DNA | variable |
MiliQ water | up to 25 µl |
Cycling conditions | |||
---|---|---|---|
initial denaturation | 98°C | 30 sec | 1x |
denaturation | 98°C | 5 - 10 sec | 25 - 30x |
annealing* | 50 – 72°C | 10 - 30 sec | 25 - 30x |
elongation** | 72°C | 20 – 30 sec/kb | 25 - 30x |
final elongation | 72°C | 2 min | 1x |
hold | 4 – 10°C |
*The annealing temperature depends on the melting temperature of the primers used.
If using LyseBlue reagent, the solution will turn colorless.
For DNA fragment sizes in the range of 200 bp to 5 kbp:
For DNA fragment sizes smaller than 200 bp or larger than 5 kbp:
Protocol
*For very small sample volumes < 30 μl adjust the volume of the reaction mixture to 50–100 μl with water.
**Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2–5 min at 70°C prior to elution.
***DNA recovery of larger fragments (> 1000 bp) can be increased by multiple elution steps with fresh buffer, heating to 70°C and incubation for 5 min.
*Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2–5 min at 70°C prior to elution.
**DNA recovery of larger fragments (> 1000 bp) can be increased by multiple elution steps with fresh buffer, heating to 70°C and incubation for 5 min.
DSM (for 1000 ml) | |
---|---|
Bacto nutrient broth (Difco) | 8 g |
10 % (w/v) KCl | 10 ml |
1.2 % (w/v) MgSO4 . 7H2O | 10 ml |
1 M NaOH | 1.5 ml |
1 M Ca(NO3)2 | 1 ml |
0.01 M MnCl2 | 1 ml |
1 mM FeSO4 | 1 ml |
Solution of 1000 ml | Beef extract | 3 g |
---|---|
Soluble starch | 10 g |
Agar | 12 g |
Distilled water | 1000 ml |
Reference:
Preparation of the 96 well plate
Photometric measurement (Plate reader – Varioskan LUX Reader, Thermo Fischer)
No. | Primer name | Sequence 5'-3' | Tm[°C] |
---|---|---|---|
1 | F-key sequence | CGGAATTCGCGGCCGCTTCTAGAGG | 67.9 |
2 | R-key sequence | GCCTGCAGCGGCCGCTACTAGTACC | 68.0 |
3 | F-message sequence | GCGTCGACGACCAAGCCTGCAAAAAC | 65.4 |
4 | R-message sequence | GCAAGCTTGTCGGTGGGTGCAATGC | 65.2 |
5 | F-qnrs1 e.coli | GCAAGCTTGAATTCGCGGCCGCTTC | 67.6 |
6 | R-qnrs1 e.coli | GCGGTACCCTGCAGCGGCCGCTACTAG | 70.9 |
7 | F-qnrs1 optimized | GCAAGCTTGAATTCGCGGCCGCTTC | 67.6 |
8 | R-qnrs1 optimized | GCGGTACCCTGCAGCGGCCGCTACTAG | 70.9 |
9 | amyE-prefix | GCGGAATTCGCGGCCGCTTCTAGATGTTTGCAAAACGATTCAAAAC | 73.6 |
10 | amyE-suffix | CGTACTAGTAGCGGCCGCTGCAGTCAATGGGGAAGAGAACCGCTTAAGC | 77.1 |
11 | F-gfp insert | GCAGATCTAAATTGAATTCAACGCTCGAATGC | 65.0 |
12 | R-gfp insert | GCGCTAGCATTACGCCAAGCTTGAATCTTGCTTG | 71.2 |
13 | F message sequencing | GCGCGTACGATCTTTCAGCCGACTC | 65.9 |
14 | R message sequencing | GCGCCGCGTTTCGGTGATGAAGAT | 65.3 |
15 | F key only amplify | GCGGACCAGCTCATGATTCTCAC | 60.9 |
16 | R key only amplify | GCGAGGAGGCTTACTTGTCTGCTTTCTTC | 65.9 |
17 | amyE int check | CTCTGCCAAGTTGTTTTGATAGAGTG | 59.2 |
18 | key only + prefix | GAATTCGCGGCCGCTTCTAGAGGACCAAGCCTGCAAAAC | 73.6 |
19 | key only + suffix | CTGCAGCGGCCGCTACTAGTAGTCGGTGGGTGCTATGGAGCGACAGAA | 78.3 |
20 | message prefix | GAATTCGCGGCCGCTTCTAGAGGACCAAGCCTGCAAAAACAAAG | 74.4 |
21 | message suffix | CTGCAGCGGCCGCTACTAGTAGTCGGTGGGTGCAATGCTTC | 76.0 |
22 | pATPI+prefix | GCGAATTCGCGGCCGCTTCTAGAGTATAGGTGAAAATGTGAACATTC | 72.9 |
23 | pATPI+suffix | CTGCAGCGGCCGCTACTAGTACAATTATCTGTCTCCTGATGAA | 73.0 |
24 | prefix sfGFP(Sp) | GAATTCGCGGCCGCTTCTAGATGTCAAAAGGAGAAGAACTTTTTAC | 71.3 |
25 | suffix sfGFP(Sp) | CTGCAGCGGCCGCTACTAGTACATTATTATTTATAAAGTTCGTCCATAC | 71.0 |
26 | VF2 | TGCCACCTGACGTCTAAGAA | 55.4 |
27 | VR | ATTACCGCCTTTGAGTGAGC | 55.4 |
28 | nucA+prefix | GAATTCGCGGCCGCTTCTAGATGAAAAAGATTTGGCTGGCGCTGGCTG | 77.4 |
29 | nucA+suffix | CTGCAGCGGCCGCTACTAGTATTATTGACCTGAATCAGCGTTGTC | 74.2 |
30 | Gb_insert_F2 | GTACATCTTTGTAACTTTATTATACGACTGGGCCTTTCGTTTTATCTGTTG | 72.0 |
*prefix and suffix are underlined