Team:Heidelberg/Sandbox
Short introduction
“The sea is everything. […] The sea is the vast reservoir of Nature. The globe began with sea, so to
speak”, Nemo said while the “Nautilus” was cruising with a school of hammerhead sharks deep
beneath the waves.
And the captain was right. Deep in the ocean billions of years ago the miracle of nature took place as
a pool of small molecules evolved to self-replicating lifeforms. The flagship role in this development
was probably taken by the most versatile class of molecules in the history of life: RNA.
Seemingly random nucleotides happened to be in the right order to form the first biocatalysts that
made life on the blue planet possible. Today we know those miracles of nature as ribozymes. Inspired
by this, humanity took evolution into their own hands to create aptamers – nucleic acids capable of
encaging molecules. This allows for the detection of virtually anything. Still this process has been
tedious and time consuming much like fishing with a rod in an ocean. We want to revolutionize this
former evolutionary process and want to make it swift like a shark tracking down its prey.
Yet to really bring out the strengths of these simple yet powerful molecules just comprised of A, U, C
and G we want to combine aptamers and ribozymes to create a toolset for the synthetic biologist to
create allosteric ribozymes able to sense a variety of molecules. Therefore, we hope to introduce the
true origins of life and the capabilities of functional RNA to iGEM.
Join us as we sail forth into new
waters of synthetic biology.
Further content
week number 38
▼2015-09-14 Test bitte löschen
10 x <i>in vitro</i> transcription buffer
50 mM Tris ph 7.5, 100 mM NaCl, 20 mM MgCl<sub>2</sub>, 0,01 % SDS
week number 37
▼2015-09-10 DNAzyme Activity: DNAzyme with ATP aptamer and calculated Kanamycin aptamer
Samples:
- 10-23 DNAzyme: xxfs032xx
- 7-18 DNAzyme: xxfs033xx
- 10-23 DNAzyme with ATP aptamer with linker: xxfs019xx
- 7-18 DNAzyme with ATP aptamer with linker: xxfs027xx
- 10-23 DNAzyme with ATP aptamer A: xxfs017xx, B: xxfs018xx
- 7-18 DNAzyme with ATP aptamer A: xxfs025, B: xxfs026xx
- 10-23 DNAzyme with calculated Kan aptamer: xxfs034xx
- 7-18 DNAzyme with calculated Kan aptamer candidate I: xxfs035xx
- 7-18 DNAzyme with calculated Kan aptamer candidate II: xxfs036xx
- 7-18 DNAzyme with calculated Kan aptamer candidate III: xxfs037xx
Stock solutions and conditions:
|
cStock |
cFinal |
|
|
Tris HCl ph 7.5 |
1 M |
50 mM |
|
DNAzyme (A) |
10 µM |
500 nM |
|
DNAzyme B |
10 µM |
500 nM |
|
Substrate |
1 µM |
200 nM |
|
NaCl |
1 M |
100 mM |
|
MgCl2 |
1 M |
20 mM |
|
SDS |
20 % |
0,01 % |
|
Adenosine in H2O:DMSO 1:2 |
33 mM |
5 mM |
|
H2O |
|
ad 25 µL |
Pipetting scheme:
|
# |
|
|
Tris HCl ph 7.5 |
DNAzyme A |
DNAzyme B |
Substrate |
NaCl |
MgCl2 |
SDS |
Adenosine |
H2O |
Final |
|
1 |
FS032 |
10-23D |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
9,50 |
25,00 |
|
2 |
FS033 |
7-18D |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
9,50 |
25,00 |
|
3 |
FS019 |
10-23DmLink |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
9,50 |
25,00 |
|
4 |
FS027 |
7-18DmLink |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
9,50 |
25,00 |
|
5 |
FS017+18 |
10-23D_A+B |
1,25 |
1,25 |
6,25 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
3,25 |
25,00 |
|
6 |
FS025+26 |
7-18D_A+B |
1,25 |
1,25 |
6,25 |
5,00 |
2,50 |
0,50 |
1,25 |
3,75 |
3,25 |
25,00 |
|
7 |
FS032 |
10-23D |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
13,25 |
25,00 |
|
8 |
FS033 |
7-18D |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
13,25 |
25,00 |
|
9 |
FS019 |
10-23DmLink |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
13,25 |
25,00 |
|
10 |
FS027 |
7-18DmLink |
1,25 |
1,25 |
0,00 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
13,25 |
25,00 |
|
11 |
FS017+18 |
10-23D_A+B |
1,25 |
1,25 |
6,25 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
7,00 |
25,00 |
|
12 |
FS025+26 |
7-18D_A+B |
1,25 |
1,25 |
6,25 |
5,00 |
2,50 |
0,50 |
1,25 |
0,00 |
7,00 |
25,00 |
|
13 |
FS032 |
-10-23D |
1,25 |
1,25 |
0 |
0 |
2,5 |
0,5 |
1,25 |
0 |
18,25 |
25,00 |
|
14 |
FS033 |
-7-18D |
1,25 |
1,25 |
0 |
0 |
2,5 |
0,5 |
1,25 |
0 |
18,25 |
25,00 |
|
15 |
FS019 |
-10-23DmLink |
1,25 |
1,25 |
0 |
0 |
2,5 |
0,5 |
1,25 |
0 |
18,25 |
25,00 |
|
16 |
FS027 |
-7-18DmLink |
1,25 |
1,25 |
0 |
0 |
2,5 |
0,5 |
1,25 |
0 |
18,25 |
25,00 |
|
17 |
FS017+18 |
-10-23D_A+B |
1,25 |
1,25 |
6,25 |
0 |
2,5 |
0,5 |
1,25 |
0 |
12 |
25,00 |
|
18 |
FS025+26 |
-7-18D_A+B |
1,25 |
1,25 |
6,25 |
0 |
2,5 |
0,5 |
1,25 |
0 |
12 |
25,00 |
|
19 |
Adenosine |
Substrate only |
1,25 |
0 |
0 |
5 |
2,5 |
0,5 |
1,25 |
3,75 |
10,75 |
25,00 |
|
# |
Tris HCl ph 7.5 |
DNAzyme A |
DNAzyme B |
Substrate |
NaCl |
MgCl2 |
SDS |
Kan |
H2O |
Final |
||
|
20 |
FS032 |
10-23D |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
21 |
FS033 |
7-18D |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
22 |
FS034 |
Kan |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
23 |
FS035 |
Kan I |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
24 |
FS036 |
Kan II |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
25 |
FS037 |
Kan III |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
12 |
25 |
|
26 |
FS032 |
10-23D |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
27 |
FS033 |
7-18D |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
28 |
FS034 |
Kan |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
29 |
FS035 |
Kan I |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
30 |
FS036 |
Kan II |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
31 |
FS037 |
Kan III |
1,25 |
1,25 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
13,25 |
25 |
|
32 |
Kan |
Substrate only |
1,25 |
0 |
0 |
5 |
2,5 |
0,5 |
1,25 |
1,25 |
13,25 |
25,00 |
|
33 |
Substrate only |
1,25 |
0 |
0 |
5 |
2,5 |
0,5 |
1,25 |
0 |
14,5 |
25,00 |
Results and Outlook:
Positive controls worked, Adenosine dependency could be detected for one candidate.
▼2015-09-10 Click reaction of Label-, Label AU RNA [1, 2, 3]
|
For 51.5µL |
Cstock |
Cfinal |
V[µL] |
|
Phosphate Buffer - pH 7, 0.1M |
100mM |
50mM |
25 |
|
Alexa 488 azide |
10µM |
400nM |
2 |
|
RNA |
1µM |
200nM |
10 |
|
CuSO4 |
20mM |
1mM |
2.5 |
|
THPTA |
50mM |
5mM |
5 |
|
NaAsc |
100mM |
1mM |
0.5 |
|
H2O |
|
|
6.5 |
- Alexa 488 azide was solved in DMSO
- Incubation at 37 °C for 12-14 hours (overnight)