In Vitro Assays
After the cloning, expression and the purification of the Fok constructs we conducted several assays to analyze the activity of the enzyme. To establish the assay and as a reference for activity we used wildtype FokI. Binding of the modified nucleotides and enzymatic activity were tested with the Fok_i / Fok_a construct.
Cutting with FokI wildtype
First the ability of FokI wildtype to cut ssDNA hybridized with a short oligonucleotide had to be confirmed. M13 ssDNA was isolated which contains four FokI cutting sites. For three of them complementary oligonucleotides were ordered with a length of 40 bases (Link zur Oligoseite) as a oligo with this length should form a helix with the M13 DNA. The M13 ssDNA was hybridized with the oligonucleotides and then incubated with FokI. We ran a agarose gel and the first gels showed a strong degredation of the ssDNA with no distinct fragment visible on the gel. But after reducing the amount of FokI and the time of incubation a clear fragment pattern showed up.
|Figure 1: Agarose gel of FokI cutting M13 ssDNA and dsDNA|
M: GeneRuler DNA Ladder Mix (Fermentas, 6 µl);
1: Untreated M13 ssDNA (300 ng);
2: Digest of M13 ssDNA with FokI and with the nucleotide Fok control 1;
3: Digest of M13 ssDNA with FokI and with the nucleotide Fok control 2;
4: Digest of M13 ssDNA with FokI and with the nucleotide Fok control 3;
5: Digest of M13 ssDNA with FokI and with the nucleotides Fok control 2 and 3;
6: Digest of M13 ssDNA with FokI and with the nucleotides Fok control 2 and 3, heat inactivation at 95°C 10 min;
7: Digest of M13 ssDNA with FokI and with the nucleotides Fok control 1 and 2;
8: Digest of M13 ssDNA without FokI and with the nucleotides Fok control 1 and 2;
9: Digest of M13 ssDNA with FokI and with the nucleotides Fok control 1 and 3;
10: Digest of M13 ssDNA with FokI and with the nucleotides Fok control 2 and 3, kept on 37°C after the hybridization;
11: Digest of M13 dsDNA with FokI.
The M13 DNA has a size of 7259 bp.
The digest of dsDNA with FokI wildtype would lead to the following
fragment pattern: 900 bp, 3560 bp, 2780 bp, 200 bp. This pattern was
visible at the gel in lane 11.
Cutting with the nucleotides Fok control 2 and 3 should create two
fragments with sizes of 6100 bp and 1100 bp. The gel picture shows two
fragments at the right size in the lanes 5 and 6 and some uncut vector
in lane 6.
Incubation with Fok control 1 and 2 should cut out a 200 bp fragment
which couldn't be seen on the gel in lane 7. Probably the DNA
concentration was to low to detect this small insert. The digest with
Fok control 1 and 3 in lane 9 didn't provide the fragments of 906 bp
and 7100 bp. Maybe the Fok control 1 nucleotide didn't work right, as
both digests with this nucleotide failed. The negative control in lane
8 didn't show any shorter fragments. The digest in lane 10 didn't work
because we forgot the DNA.
Hybridize M13 ssDNA with Fok control oligos 2 and 3
|10 µl||M13 ssDNA (c = 152 ng/µl, 02.09.09, sample #3 + 4)|
|5 µl||MgCl2 (5 mM)|
|5 µl||Tris-HCl pH 8 (100 mM)|
|2 µl||Fok control 2 (10 ng/µl; 1 µM)|
|2 µl||Fok control 3 (10 ng/µl; 1 µM)|
The hybridization was also done with just Fok control 1, 2 or 3 and
with Fok control 1 + 3 and 1 + 2.
Incubation with the thermocycler and the program ORIGAMI0 (heat to 95°C and cool down slowly to 37°C over 1 hour).
10 µl of the hybridized DNA were used for a cutting
with 1,2 µl buffer 4 (NEB) and 0.5 µl FokI
(wildtype Fok from NEB, 4000 units/ml). The
digest was incubated for 30 minutes at 37°C.
We also tested whether the construct binds specifically to the
tagged oligonucleotides. To analyse this, a initial fluorescence
quenching experiment was conducted. The fluorescein which is bound to
the nucleotide is excited at about 500 nm and emits at about 520 nm.
When the protein binds, the fluorescence is quenched.
Protein - Fluorescein - Interaction
The fluorescence was measured with a NanoDrop 3300 Fluorospectrometer. The excitation maximum of the blue LED is at 470 nm. For each measurement a full emission spectrum from 510 nm to 800 nm was recorded.
Fixed protein concentration in the assay: 40 nM His-FluA-SplitLinker-Fok_i
Variable oligo concentrations in the assay: 3,3 µM; 0,33 µM and 0,033 µM monoligo A
|Figure 2: Initial fluorescence measurement with the NanoDrop 3300|
Cutting with the Fok_a /Fok_i construct
The first approach was to use a cytosolic solution containing the Fok_a and Fok_i proteins to cut a Cy3-tagged 80 bp oligonucleotide. Therefore co-transformed XL1Blue cells with both constructs (for details: in vivo assay) were incubated till they reached an OD600 of 2 and then centrifuged, resuspended and sonicated. The 80 bp oligo was hybridised with the monoligo A and monoligo B and afterwards incubated for one hour either directly with the sonicated cell suspension or with a centrifuged cell solution.
|Figure 3: Scheme of the cutting assay|
After the digestion of the Cy3-tagged 80 bp oligonucleotide two 40 bp fragments should be visible. As the gel pictures showed the untreated 80 bp nucleotide emitted very strong. The digested samples didn't have any 80 bp fragment left and a distinct fragment with a shorter size of about 40 bp was visible on the agarose gel.
Hybridize Cy3-tagged nucleotide with monoligo A and B
|Sample 1||Sample 2|
|4 µl 100 mM Tris-HCl pH 8||4 µl 100 mM Tris-HCl pH 8|
|8 µl 5 mM MgCl2||8 µl 5 mM MgCl2|
|5 µl Cy3-80mer nucleotide (c = 2500 ng/µl, 100 µM)||5 µl Cy3-80mer nucleotide (c = 2500 ng/µl, 100 µM)|
|8 µl fluo-mono-oligo-dig (100 µM)||6 µl diAB1_fluo-oligo (100 µM) and 6 µl diB1_dig-oligo (100 µM)|
|15 µl water||11 µl water|
|1. 96°C -> 7 min.|
|2. 96°C -> 1 min.//-1°C R=0.3°C/sec|
|3. goto 2 repeat 70|
|4. hold 4°C|
1. overnight culture of double transformed XL1 blue cells in DYT Amp/Chlor @24 °C
2. Next day inoculated 15 ml DYT ampicilin/chloramphenicol with 100 µl overnight culture
3. Shake at 24°C until OD600 = 1
4. Induced protein expression with 0.6 mM IPTG
5. Shake at 24°C for 1 hour
6. Harvested cells by centrifugation at 5000xg for 15 min. at 4°C
7. Resuspended pellet in 2 ml P1
a. P1: a. 50 mM Tris, 1M NaCl, pH 7,5, protease inhibitor coctail PIC 43mg/20ml culture, RnaseI 10µg/µl --> 4µl
8. Sonicated 5 min. on ice
9. 100 µl aliquot for crude cell lysate cutting assay
10. Centrifuged remaining lysate at 4°C, 16000xg and 10 min. for cleared cell lysate cutting assay
11. Cutting assay:
2. 12,8 µl crude cell lysate
3. 2 µl 10x Buffer Fok (200 mM Tris-acetate, 500 mM potassium acetate, 100 mM Magnesium Acetate, 10 mM Dithiothreitol, pH 7.9 @ 25°C)
4. 0,2 µl 100x BSA
2. 95 °C 4 min --> melt cut strands
3. 89 °C hold --> add 1 µl formamide & 3µl gel loading dye
4. load onto 3 % agarose gel