The biological realization of CryptoGErM consisted of three
subprojects: integration, decoy and key deletion.
Integration
The first subproject is the integration, the key and message
sequences were integrated into the genomic DNA of two separate
Bacillus subtilis strains. In order to achieve this we had the key
and the message sequence synthesized by IDT. Then we cloned it in
pSB1C3 to amplify it from there and cloned it in the B. subtilis
integration biobrick BBa_K823023. This vector can be used to
integrate sequences into the amyE locus of Bacillus subtilis. From
there we integrated it in the genome of B. subtilis 168 trp+. For
message and key transmission these B. subtilis strains were
sporulated.
The location in the genomic DNA makes it harder to retrieve the
data, since whole genome sequencing is required. The message is
protected by a digital lock: the key, and thus doesn’t need further
biological protection.
The key is not encrypted and has to be protected by a biological
lock. We followed different approaches to design a multi-layered
biological lock.
We followed two main ideas, namely hiding the key and deleting
the key when unauthorized parties handle it.
Decoy
Hiding the key became called the decoy approach.The
key-containing spore will be send in a mixture of different decoy
spores. The recipient has to be aware of the special treatment that
is required to select the correct spores from the decoy. We have
been working on a photoswitchable ciprofloxacin compound.
Only the knowledge about the right wavelength allows the
recipient to activate the added ciprofloxacin and thus start
selection of the right spore strain.
Our design of the decoy approach included the biobricks for
ciprofloxacin resistance, a super folder GFP and a pATP promotor.
Key deletion
Another biological security layer is provided by our key
deletion system. We have been working on two different approaches.
The first is a nucA killswitch which is made out of an assembly of
different biobricks. Atc or tetracycline have to be added to
inhibit the tetR promoter to stop the expression of the nucA and
digestion of the key sequence.
The second approach makes use of a CRISPRcas system which will
delete the key from the genome if no special treatment is applied.
Addition of Atc or tetracycline will stop the cas9 expression.
This system is highly flexible and biological layers can easily
be added or modified to the wishes of the user.
Proof of concept
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).
Figure 1: CryptoGErM (B. subtilis) colonies after
being transformed with either the message or the key K823023
plasmid.
Figure 2 Starch test for the B. subtilis colonies
potentially carrying message or key.
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.
Figure 3. (A) B. subtilis spores carrying the
message under the phase contrast microscope. (B) CryptoGErM
(left, with sunglasses) carrying the message and (right, cute)
carrying the key.
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).
Figure 4 (A) Spores of Bacillus subtilis carrying
our encrypted message during the sending simulation. (B)
Colonies obtained from germinating the spores on LB agar.
Figure 5.Colony PCR on colonies obtained from Bacillus subtilis. Primers used: (o) F/R message sequence. Product 572 bp. (n) F/R message sequencing. Message product 916 bp. For the key the F/R message sequencing primers were used. Key product 178 bp.
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).
Figure 6. (A) PCR product, amplified message
sequence, on the way to be sequenced by Macrogen. (B)
Chromatogram of the sequencing result. (C) Obtained sequence.
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.
.
Figure 7. Decryption program with the message
sequence
The next step was to actually send the message to another iGEM
team to demonstrate the functionality under real world conditions. Therefore read Collaborations.
Figure 8. (A) Members from iGEM team Eindhoven 2016 decrypting our message sent in spores. (B) Also the iGEM team Wageningen is happy to decrypt our message. Blue arrow indicates decrypred message
Decoy
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]!
Experiment setup
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
Table 1. The initial ratio of mutant vs wild-type
strains was screened from 1 to 10 million. The final ratio was
measured as the relative (green:gray) area under the curve (AUC)
obtained in the flow cytometer (Figures 2, 4, 6, 8, 10, 12, 14, 16,
18 and 20). [Using: Flowing Software 2.5]
Results
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.
Figure 2 & 3: Wildtype without spectinomycin
Figure 4 & 5: sfGFP strain without spectinomyin
Figure 6 & 7: Wildtype with spectinomycin
Figure 8 & 9: sfGFP strain with spectinomyin
Figure 10 & 11: 1:1 without spectinomycin
Figure 12 & 13: 1:1 with spectinomycin
Figure 14 & 15: 1:150 without spectinomycin
Figure 16 & 17: 1:150 with spectinomycin
Figure 18 & 19: 1:10.000.000 without spectinomycin
Figure 20 & 21: 1:10.000.000 with spectinomycin
Discussion
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.
Conclusion
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.
References:
[1]Fox EJ, Reid-Bayliss KS,
Emond MJ, Loeb LA (2014) Accuracy of Next Generation Sequencing
Platforms. Next Generat Sequenc & Applic 1: 106.
doi:10.4172/jngsa.1000106
[2]Pochon (2013). Evaluating
detection limits of next-generation sequencing for the surveillance
and monitoring of international marine pests. PLos One
8(9):e73935
[3]Schmitt MW, Kennedy SR,
Salk JJ, Fox EJ, Hiatt JB, et al. (2012) Detection of ultra-rare
mutations by next-generation sequencing. Proc Natl Acad Sci U S A
109: 14508-14513.
Labjournal
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.
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.
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.
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:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
30s
(30X)
72ºC
2:00 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.
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.
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:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
1:30 min
(30X)
72ºC
2:00 min
10ºC on hold
DNA electrophoresis:
For detailed information on how to prepare and run agarose 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.
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.
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.
The message sequence was ordered as a gBlock from IDT to construct the message B.
subtilis strain. In a first approach this was done with the pDR111 B.
subtilis integration plasmid, which can also be amplified by E. coli.
Therefore the first step was to clone the message sequence into the pDR111 in E. coli. pDR111 is an integration plasmid which can be integrated into the B. subtilis genome.
By double cross-over it replaces amyE gene, which is necessary for production of alpha-amylase
(see Subtiwiki.uni-goettingen.de) with desired insert which is located between amyE front flanking region and amyE back flanking region. See the plasmid map below. We used this plasmid for integration of our message sequence into the B. subtilis 168 sub+. The other approach made use of the BioBrick integration plasmid BBa_K823023 (Message sequence in BBa_K823023).
PCR
Experiment:
25/07/16: The message sequence was amplified from gBlock ordered from IDT (Integrated DNA technologies).Primers used for the amplification were F-message sequence and R-message sequence (primer sequences can be found here).
PCR mixture:
50 μl PCR assay was performed according to the following
protocol.
Figure 2. DNA electrophoresis with amplified message sequence 572 bp in size.
Conclusion:
PCR of the message sequence from the IDT gBlock was successful. It was verified by DNA electrophoresis.
Correct size of a band could be seen and that was 572 bp.
Procedure after gel validation:
PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience
(see protocol).
Restriction digestion
Experiment:
26/07/16: On this day restriction digestion of PCR product of the message sequence and pDR111 integration plasmid was done. Message sequence as an insert was cut with SalI and HindIII restriction enzymes. Integration plasmid pDR111 as a vector was cut with exactly same restriction enzymes.
RD mixture:
20 μl RD assay was performed according to the following
protocol.
Figure 3. DNA electrophoresis with samples from restriction digestion.
Conclusion:
RD of the PCR product of message sequence and integration plasmid pDR111 was successful. It was verified by DNA electrophoresis. Correct size of bands could be seen and that was 7,834 bp for pDR111 and 572 bp for message sequence.
Procedure after gel validation:
Digested samples were cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin)
(see protocol).
Ligation
Experiment:
26/07/16: Restriction digestion was followed by overnight ligation. Vector (integration plasmid pDR111) and insert (message sequence) was ligated in 1:5 molar ratio.
Ligation mixture:
20 μl ligation assay was performed according to the following
protocol.
Transformation
Experiment:
27/07/16: This day transformation into E. coli Top10 and MC1061 cells was done. Correct clones were selected on 100 μg/ml ampicillin plates. Transformation protocol used can be found here.
28/07/16: Next day 12 colonies were obtained on the plates of E. coli MC1061 strain. To verify correct transformants colony PCR was performed (see Colony PCR protocol). Primers F-message sequence and R-message sequence were used for this colony PCR. Sequences of these primers can be found here.
PCR mixture:
25 μl PCR assay was performed according to the following
protocol.
PCR set-up:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
1:30 min
(30X)
72ºC
2:00 min
10ºC on hold
DNA electrophoresis:
For detailed information on how to prepare and run agarose gel see following protocol.
Figure 4. DNA electrophoresis with samples from colony PCR.Figure 5. Transformation of pDR111+message into E. coli MC1061.
Conclusion:
Transformation of pDR111+message into E. coli MC1061 appeared to be successful. Transformation efficiency was low, only 12 colonies were obtained, but 4 colonies seemed to be correct clones. And those were colonies 1, 8, 10 and 11 (see Figure 5). Correct sizes of the bands were obtained: 572 bp. Those colonies were grown overnight (see cell culture protocol) from pre-glycerol stocks made for colony PCR (see colony PCR protocol).
Validation
Experiment:
29/07/16: Grown cultures of E. coli MC1061 with pDR111+message were taken out from the incubator after overnight incubation. Glycerol stocks) were made and plasmid isolation was performed (see Mini prep protocol).
Firstly, concentration of the plasmids was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.
Sequencing:
Plasmids pDR111+message from colonies 1, 8, 10 and 11 were sent for sequencing)
with the sequencing primer Gb_insert_F2 ((see primer list)).
Conclusion:
Sequencing results showed that cloning of message sequence into integration plasmid pDR111 was successful. However just sequencing result 249 (Figure 9) has 100 % homology when compared to the reference (message sequence). Others have some bases missing or bases are not matching, it could be due to faulty sequencing. See sequencing results below.
Figure 6. Sequencing result 246 of the message in pDR111.Figure 7. Sequencing result 247 of the message in pDR111.Figure 8. Sequencing result 248 of the message in pDR111.Figure 9. Sequencing result 249 of the message in pDR111.
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.
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.
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.
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.
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:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
2:00 min
(30X)
72ºC
2:00 min
10ºC on hold
DNA Electrophoresis:
For detailed information on how to prepare and run agarose gels see
following protocol.
Figure 1. DNA electrophoresis with amplified message
sequence (M1 and M2) 599 bp in size. C - is water control.
Conclusion:
Amplification of the message sequence and addition of the prefix and
suffix to this sequence was successful. It was verified by DNA
electrophoresis (see above). Correct size of a band could be seen and
that was 599 bp.
07/10/16: On this day restriction digestion of PCR product of the
message sequence and BBa_K823023 was done. Message sequence
as an insert was cut with EcoRI and PstI restriction enzymes.
BBa_K823023 as a vector was cut with exactly same restriction
enzymes.
RD mixture:
20 μl RD assay was performed according to the following protocol.
DNA Electrophoresis:
For detailed information on how to prepare and run
agarose gels see following protocol.
Figure 2. DNA electrophoresis with cut backbone
BBa_K823023.
Conclusion:
RD of the PCR product of message sequence and BBa_K823023 was
successful. RD of BBa_K823023 was verified by DNA electrophoresis. Correct size of band could be seen and that was ∼6000 bp.
Procedure after gel validation:
The upper band was cut out from the gel and DNA was extracted by Agarose gel
extraction kit (Jena Bioscience).
Ligation
Experiment:
07/10/16 Restriction digestion was followed by overnight
ligation. Vector (BBa_K823023) and insert (message sequence) was
ligated in 4:6 molar ratio.
Ligation mixture:
20 μl ligation assay was performed according to the following
protocol.
Transformation
Experiment:
08/10/16: On this day transformation into E. coli Top10 was done.
Selection was made on 100 μg/ml ampicillin LB agar plates.
Transformation protocol used can be found here.
09/10/16: Next day we could assume that our transformation was
successful -> colonies were obtained on the plates of E. coli Top10
strain. To verify correct transformants colony PCR was
performed (see colony PCR protocol). Primers message
prefix and message suffix were used for this colony PCR. Sequences of
these primers can be found here.
PCR mixture:
50 μl PCR assay was performed according to the following protocol.
Figure 3. DNA electrophoresis with samples from colony
PCR.Figure 4. Transformation of BBa_K823023+message into
E. coli Top10.
Conclusion:
Transformation of BBa_K823023+message into E. coli Top10 appeared to
be successful. Correct sizes of the bands from colony PCR (see Figure
3) were obtained: 599 bp. Correct clones were grown overnight (see cell
culture protocol) from pre-glycerol stocks
made for colony PCR (see colony PCR protocol.
Validation
Experiment:
10/10/16 Grown cultures of E. coli Top10 with BBa_K823023+message were
taken out from the incubator after overnight incubation. Glycerol stocks were made and plasmid isolation was
performed (see (Fast-n-Easy Plasmid Mini-Prep Kit - Jena Bioscience) protocol). Concentration of the
plasmids obtained was measured on Nanodrop and plasmids were stored at
-20°C.
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.
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.
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:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
1:30 min
(30X)
72ºC
2:00 min
10ºC on hold
DNA electrophoresis:
For detailed information on how to prepare and run agarose gel see
following protocol.
Figure 4. DNA electrophoresis 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.
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.
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.
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.
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.
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.
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.
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:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
1:30 min
(30X)
72ºC
2:00 min
10ºC on hold
DNA electrophoresis:
For detailed information on how to prepare and run agarose gel see
following protocol.
Figure 4. 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!
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 qnrS1E. 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:
95ºC
2:00 min
95ºC
30s
(12X)
60ºC
30s
(12X)
72ºC
1:30 min
(12X)
72ºC
2:00 min
10ºC on hold
DNA Electrophoresis:
For detailed information on how to prepare and run agarose gels see
following protocol.
Figure 1. 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.
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.
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.
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 ).
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:
95ºC
2:00 min
95ºC
30s
(12X)
60ºC
30s
(12X)
72ºC
1:30 min
(12X)
72ºC
2:00 min
10ºC on hold
DNA Electrophoresis:
For detailed information on how to prepare and run agarose gels see
following protocol.
Figure 1. DNA electrophoresis 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.
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).
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.
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).
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.
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:
95ºC
2:00 min
95ºC
30s
(30X)
60ºC
30s
(30X)
72ºC
30s
(30X)
72ºC
2:00 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.
Protocols
All protocols which were used throughout our project can
be found below or downloaded here.
Transformation of B. subtilis
Day 1
Streak out desired strain and incubate the plate overnight at 37°C.
Day 2
Pick a nice big colony and drop it in 2 ml of completed 1X MC medium (see below).
Grow at 37°C for 5 hours (or more if culture is not really turbid).
Mix 400 µl of culture with DNA (usually 1 µg) in fresh tube.
Grow for an additional 2 hours at 37°C.
Plate on selective antibiotic plates.
Incubate overnight at 37°C.
Preparation of MC medium
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)
Mix everything in 40-50 ml H2O.
Then adjust to 100 ml.
Filter sterilize.
Freeze at -20°C.
Transformation of E. coli (Standard protocol)
Add ligation mixture to the tube of competent cells.
Leave 30 min on ice.
Heat shock for 5 min at 37°C or for 45 sec at 42°C.
Add 300 µl of LB medium.
Place at 37°C for 30 – 60 min. Shake vigorously (220 rpm).
Plate 30 µl on 1 selection plate, and 300 µl on another.
Transformation of E. coli (NEB® 5-alpha Competent E. coli High Efficiency Transformation Protocol)
Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear.
Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
Add 1 – 5 µl containing 1 pg – 100 ng of plasmid DNA to the cell mixture.
Carefully flick the tube 4 – 5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 min. Do not mix.
Heat shock at exactly 42°C for exactly 30 sec. Do not mix.
Place on ice for 5 min. Do not mix.
Pipette 950 µl of room temperature SOC into the mixture.
Place at 37°C for 60 min. Shake vigorously (220 rpm) or rotate.
Warm selection plates to 37°C.
Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
Spread 50 – 100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
Alternatively, incubate at 30°C for 24 – 36 hours or 25°C for 48 hours.
Reagents supplied:
6 x 0.2 ml/tube of chemically competent NEB 5-alpha Competent E. coli cells
25 ml of SOC Outgrowth Medium
0.025 ml of 50 pg/µl pUC19 Control DNA
Calcium Chloride Competent cells
Label 28 sterile 1.5 ml tubes with name of E. coli strain
All solutions, glass pipettes, pipette tips, 50 ml tubes and 1.5 ml tubes must be sterile and pre-cooled
All work must be done quickly and ON ICE (fresh ice, not half melted!)
Day 1
Streak E. coli strain on LB agar plate and incubate overnight at 37°C.
Day 2
Pick one colony from fresh agar plate and inoculate 3 ml of LB.
Incubate overnight at 37°C and 220 rpm.
Day 3
Inoculate LB medium with 1:100 overnight culture (i.e. 1 ml for 100 ml LB) in 500 ml flask.
Harvest the bacterial cell culture (1 – 3 ml) by centrifugation.
Resuspend pelleted bacterial in 300 µl Lysis Buffer by pipetting or vortexing for 1 min.
Add 300 µl of Neutralization Buffer (containing RNase A) to sample.
Mix it gently by inverting the tube 4 – 6 times (do not vortex).
Centrifuge at 10,000 g for 5 min at room temperature in a micro-centrifuge.
The color of the binding mixture should change to bright yellow indicating a pH of 7.5 required for optimal DNA binding.
Place a Binding Column into a 2 ml collection tube.
Add 100 µl of Activation buffer into the Binding Column.
Centrifuge at 10,000 g for 30 sec in a micro-centrifuge.
Apply the supernatant from steps 3 – 5 into the activated Binding Column by decanting or pipetting.
Centrifuge at 10,000 g for 30 sec.
Discard the flow-through.
Place the DNA loaded Binding Column into the used 2 ml tube.
Apply 500 µl of Washing Buffer (containing Ethanol) to the Binding Column.
Centrifuge at 10,000 g for 30 sec and discard the flow-through.
Optional Secondary Washing (Recommended only for DNA >200 bp, if highly purified DNA is required).
Add 700 µl of Washing Buffer to the Binding Column.
Centrifuge at 10,000 g for 30 sec and discard the flow-through.
Centrifuge again for 2 min to remove residual Washing Buffer.
Place the Binding Column into a clean 1.5 ml tube (not provided in the kit).
Add 30 – 50 µl Elution Buffer or dd-water to the center of the column membrane.
Incubate for 1 min at room temperature.
Centrifuge at 10,000 g for 1 min to elute DNA.
Plasmid mini-prep (QIAprep® Spin Miniprep Kit)
Pellet 1 – 5 ml bacterial overnight culture by centrifugation at 8000 rpm (6800 x g) for 3 min at room temperature (15 – 25°C).
Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-centrifuge tube.
Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4 – 6 times until the solution becomes clear.
Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4 – 6 times.
If using LyseBlue reagent, the solution will turn colorless.
Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table top micro-centrifuge.
Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting.
Centrifuge for 30 – 60 sec at 13,000 rpm (~17,900 x g) and discard the flow-through.
Recommended:
Wash the QIAprep spin column by adding 500 µl Buffer PB.
Centrifuge for 30 – 60 sec at 13,000 rpm (~17,900 x g) and discard the flow-through.
Note: This step is only required when using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content.
Wash the QIAprep spin column by adding 750 µl Buffer PE.
Centrifuge for 30 – 60 sec at 13,000 rpm (~17,900 x g) and discard the flow-through.
Centrifuge for 1 min at 13,000 rpm (~17,900 x g) to remove residual wash buffer.
Place the QIAprep column in a clean 1.5 ml micro-centrifuge tube.
To elute DNA, add 50 µl Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the QIAprep spin column.
Let it stand for 1 min, and centrifuge for 1 min at 13,000 rpm (~17,900 x g).
DNA Clean-up (PCR Purification Kit – Jena Bioscience)
For DNA fragment sizes in the range of 200 bp to 5 kbp:
Add 5 volumes of Binding Buffer to 1 volume of DNA sample and mix well.
For example, if the volume of your DNA sample is 50 µl, add 250 µl Binding Buffer.
For DNA fragment sizes smaller than 200 bp or larger than 5 kbp:
Add 3 volumes Binding Buffer and 2 volumes of Isopropanol to the PCR sample.
For example, if the volume of your DNA sample is 50 µl, add 150 µl Binding Buffer and 100 µl Isopropanol.
Protocol
Place a Spin Column into a 2 ml collection tube.
Add 100 µl of Activation Buffer into the Spin Column.
Centrifuge at 10,000 g for 30 sec in a micro-centrifuge.
Apply the sample mixture from step A or B into the activated Spin Column.
Centrifuge at 10,000 g for 30 sec in a micro-centrifuge.
Discard the flow-through.
Place the DNA loaded Spin Colum into the used 2 ml tube.
Apply 700 µl of Washing Buffer to the Spin Column.
Centrifuge at 10,000 g for 30 sec and discard the flow-through.
Optional Secondary Washing (Recommended only for DNA >200 bp, if highly purified DNA is required).
Add 700 µl of Washing Buffer to the Binding Column.
Centrifuge at 10,000 g for 30 sec and discard the flow-through.
Centrifuge again for 2 min to remove residual Washing Buffer.
Place the Binding Column into a clean 1.5 ml tube (not provided in the kit).
Add 30 – 50 µl Elution Buffer or dd-water to the center of the column membrane.
Incubate for 1 min at room temperature.
Centrifuge at 10,000 g for 1 min to elute DNA.
DNA Clean-up (NucleoSpin® Gel and PCR Clean-up)
Mix 1 volume of sample with 2 volumes of Buffer NTI (e.g. mix 100 μl PCR reaction and 200 μl Buffer NTI)*.
Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 ml) and load up to 700 μl sample.
Centrifuge for 30 sec at 11,000 x g.
Discard flow-through and place the column back into the collection tube.
Load remaining sample if necessary and repeat the centrifugation step.
Add 700 μl Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column.
Centrifuge for 30 sec at 11,000 x g.
Discard flow-through and place the column back into the collection tube.
Recommended: Repeat previous washing step to minimize chaotropic salt carry-over and improve A260/A230 values.
Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube**.
Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 ml microcentrifuge tube (not provided).
Add 15–30 μl Buffer NE***.
Incubate at room temperature (18–25°C) for 1 min.
Centrifuge for 1 min at 11,000 x g.
*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.
Gel extraction (Agarose Gel Extraction Kit – Jena Bioscience)
Cut the area of gel containing the DNA fragment.
Transfer the excised gel to a clean 1.5 ml microtube.
Add 3 volumes of Extraction Buffer to 1 volume of the sliced gel. For example, add 300 µl Extraction Buffer to each 100 mg (approx. 100 µl) gel. For gels containing >2.5 % agarose, add 6 volumes of Extraction Buffer per gel volume.
Incubate at 60 °C for 10 min with occasional mixing to ensure gel dissolution.
For DNA fragment sizes smaller than 200 bp or larger than 5 kbp and to enhance yield add 1 volume Isopropanol per gel volume to the dissolved gel and mix well.
Place a Spin Column into a 2 ml collection tube.
Add 100 µl of Activation Buffer into the Spin Column.
Centrifuge at 10,000 g for 30 sec in a micro-centrifuge.
Apply the sample mixture from steps 3 (5), 4 into the activated Spin Column.
Centrifuge at 10,000 g for 30 sec in a micro-centrifuge.
Discard the flow-through.
Place the DNA loaded Spin Colum into the used 2 ml tube.
Apply 700 µl of Washing Buffer to the Spin Column.
Centrifuge at 10,000 g for 30 sec and discard the flow-through.
Optional Secondary Washing (Recommended only for DNA >200 bp, if highly purified DNA is required).
Add 700 µl of Washing Buffer to the Binding Column.
Centrifuge at 10,000 g for 30 sec and discard the flow-through.
Centrifuge again for 2 min to remove residual Washing Buffer.
Place the Spin Column into a new 1.5 ml microcentrifuge tube (not provided).
Add 30 – 50 µl Elution Buffer or dd-water to the center of the column membrane.
Incubate for 1 min at room temperature.
Centrifuge at 10,000 g for 1 min to elute DNA.
Gel extraction (NucleoSpin® Gel and PCR Clean-up)
Take a clean scalpel to excise the DNA fragment from an agarose gel. Remove all excess agarose.
Determine the weight of the gel slice and transfer it to a clean tube.
For each 100 mg of agarose gel < 2 % add 200 μl Buffer NTI. (For gels containing > 2 % agarose, double the volume of Buffer NTI).
Incubate sample for 5–10 min at 50°C. Vortex the sample briefly every 2–3 min until the gel slice is completely dissolved.
Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 ml) and load up to 700 μl sample.
Centrifuge for 30 sec at 11,000 x g.
Discard flow-through and place the column back into the collection tube.
Load remaining sample if necessary and repeat the centrifugation step.
Add 700 μl Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column.
Centrifuge for 30 sec at 11,000 x g.
Discard flow-through and place the column back into the collection tube.
Recommended: Repeat previous washing step to minimize chaotropic salt carry-over and low A260/A230 values.
Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube*.
Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 ml microcentrifuge tube (not provided).
Add 15–30 μl Buffer NE**.
Incubate at room temperature (18–25°C) for 1 min.
Centrifuge for 1 min at 11,000 x g.
*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.
Preparation of the spore stock of B. subtilis
Inoculate 20 ml of LB medium with cells from a fresh colony of B. subtilis . Always use flasks that comprise at least 5x times the volume of media used, and always use lids that are able to allow air passage.
Culture for 6 – 8 hours at 37°C with shaking at 200 rpm. B. subtilis grows best at 37°C and has a doubling time of 30 min.
Dilute 1:200 into 1000 ml DSM medium in a sterile 1 liter flask and grow at 37°C with shaking at 200 rpm. Add 5 ml of the culture to 1000 ml medium. Check samples for spores daily using phase contrast microscopy (see Phase Contrast Microscopy Protocol). Optional use of Gram stain (see Gram staining protocol) to distinguish between vegetative cells (purple) and spores (transparent).
After 2 – 3 days >90% of the population should have sporulated.
Pellet cells at 9000 rpm for 20 min (keeping temperatures low), otherwise for small quantities a benchtop centrifuge will suffice.
Wash spores with ice-cold water 8 – 10 times to remove residual nutrients and lyse remaining vegetative cells. (Resuspend pellets in water, centrifuge, discard supernatant, repeat).
Store spores at -20°C for long-term storage or at 4°C, with weekly changes of water.
Difco Sporulation Medium (DSM)
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
Bring pH to 7.6.
Filter sterilize.
Add the following sterile (autoclaved) component solutions to 1000 ml of the cooled DSM medium prior to use:
1 M Ca(NO3)2
1 ml
0.01 M MnCl2
1 ml
1 mM FeSO4
1 ml
Integration check: Starch test
Inoculation: Use a fresh (16- to 18-hour) pure culture of test bacteria as an inoculation source. Pick a single isolated colony and either single streak or spot inoculate the surface of the agar medium.
Incubation: Incubate plates overnight at 37°C.
Starch Hydrolysis Test: After proper inoculation and incubation, flood the surface of the agar with Gram’s iodine solution. Record results immediately as the blue color formed with starch may fade giving a false-positive result of absence of starch.
Appearance of a clear zone surrounding the bacterial growth indicates starch hydrolysis (+) by the organism due to its production of the extracellular enzymes. The clear zone will start out yellow (from the iodine) and becomes progressively lighter yellow and then clear -> indicates wrong clones.
A blue/black or purple zone surrounding the growth indicates that starch is present and has not been hydrolyzed (-) and the organism did not produce the extracellular enzymes -> indicates right clones.
Starch plates
Solution of 1000 ml
Beef extract
3 g
Soluble starch
10 g
Agar
12 g
Distilled water
1000 ml
Suspend the first three ingredients in 1000 ml of distilled water. Mix thoroughly.
Heat with frequent agitation and carefully bring to just boiling (excessive boiling may hydrolyze starch).
Autoclave.
After sterilization pour the melted medium into sterilized petri plates (approximately 20-30 ml per plate) and let it solidify before use.
IDT gBlocks®
Resuspending gBlocks Gene Fragment
Centrifuge the tube for 3 – 5 sec at a minimum of 3000 x g to ensure the material is in the bottom of the tube.
Add 1X TE to reach a final concentration of 10 ng/µl.
Vortex briefly.
Incubate at 50°C for 20 min.
Briefly vortex and centrifuge.
Amplifying gBlocks Gene Fragment
For gBlocks Gene Fragments ≤ 1kb, amplification can be performed using a high fidelity polymerase.
To avoid sequence mutations due to amplification errors, limit cycle to 12 or fewer.
For gBlocks Gene Fragments > 1kb, we do not recommend amplification.
Time-lapse microscopy/Phase-contrast microscopy
Preparation of the 1.5% agarose
Measure out 150 mg of agarose (for 1.5 % agarose) and add either 10 ml of LB medium (time-lapse) or 1X TBE buffer (phase-contrast images).
Dissolve the agarose.
Store at 55 - 60°C for repetitive use.
Preparation of the cell cultures
Pick a single colony from the plate or from glycerol stock.
Put the colony in a 15 ml sterile tube and add 3 ml of LB medium.
(If needed, add selective antibiotics to the medium before adding a single colony).
Clean two microscope glass slides with 70 % ethanol and water.
Take a gene frame and carefully remove one of the plastic foils from the gene frame without causing disassembly of the plastic cover on the other side of the gene frame.
Attach the gene frame in the middle of one of the glass slides by first facilitating contact on just one side, followed by guided attachment of the remaining gene frame with a fingernail. Prevent air bubbles while attaching the gene frame to the glass slide.
Transfer 500 μl of the warm agarose-LB or agarose-TBE in the middle of the gene frame. Make sure the whole area including (the borders) is fully covered.
The following steps have to be carried out quickly to prevent excessive drying of the agarose-LB or agarose-TBE.
Place the second glass slide on the agarose-LB or agarose-TBE filled gene frame. Try to avoid air bubbles. Place the sandwiched slides in a horizontal position for 45 min at 4°C in the refrigerator to allow the agarose-LB or agarose-TBE to solidify sufficiently.
Carefully slide off the upper glass slide. Use a razor blade to cut out agar strips of ~5 mm width within the gene frame, on which the cells will be grown.
Carefully remove the second and final plastic cover from the gene frame to expose the sticky side of the gene frame.
Load single cells on the solid medium without touching it with the pipet tip. Use 2.5 μl for a whole strip, or 1 μl for a small square. Always start on top of the agarose pad and allow the liquid to disperse equally on its assigned growth area by turning the slide up and down. The slide is ready, as soon as the edges of the liquid become corrugated and movement of the liquid is no longer visible when turning the slide.
Place a clean microscope slide cover slip on the gene frame from one side to the other (avoid air bubbles). Assure complete attachment by applying pressure on the cover slip along the gene frame with your fingernail. If the cover slip is placed on the cells without allowing them to dry long enough, cells tend to grow on top of each other during the experiment. Also be careful not to wait too long before applying the cover slip, since the agarose will then be too dry.
