Team:Stockholm/9 September 2010
From 2010.igem.org
Contents |
Andreas
Cloning of N-CPPs into pSB1C3
Since we realized that the method we used for cloning the N-CPPs can cause also the intervening sequences to insert into pSB1C3, I decided to redo some clonings. Since the intervening sequences were designed with unique restriction sites, digestion with these endonucleases should prevent cloning of these.
Digestion of N-CPP cluster
[N-CPP plasmid] = 672 ng/μl
Tested the FastDigest buffer, even though conventional Fermentas restriction enzymes were used.
N-CPP | |
---|---|
1st incubation | |
10X FastDigest buffer | 3 |
DNA (2 μg) | 3 |
dH2O | 19 |
XbaI (conv.) | 1 |
AgeI (conv.) | 1 |
27 μl | |
2nd incubation | |
FD BamHI | 1 |
FD HindIII | 1 |
29 μl |
- 1st incubation: 37 °C, 2:30
- 2nd incubation: 37 °C, 0:30
- Inactivation: 80 °C, 20 min
Ligation
Two ligation reactions were prepared to test the efficiency of two different ligation buffers.
- Vector: Dig pSB1C3 X+A EXTR (13.72 ng/μl)
- Insert: Dig N-CPP X+A 9/9 (31.25 ng/μl)
Lig pSB1C3 NCPP 1 9/9 | Lig pSB1C3 NCPP 2 9/9 | |
---|---|---|
5X Rapid Ligation buf. | 4 | 0 |
10X T4 DNA ligase buf. | 0 | 2 |
Vector DNA | 4 | 4 |
Insert DNA | 11 | 11 |
dH2O | 0 | 2 |
T4 DNA ligase | 1 | 1 |
20 μl | 20 μl |
- Incubation: 22 °C, 16 min
Digestion of previous ligation sample
- Ligation mix: Lig pSB1C3.N-CPP* 6/9
Ligation mix | 15 |
10X FD buffer | 2 |
FD BamHI | 1 |
FD HindIII | 1 |
19 μl |
---|
- Incubation: 37 °C, 30 min
- Inactivation: 80 °C, 15 min
Transformations
Standard transformation protocol.
- 3 μl ligation mix
- Lig pSB1C3.N-CPP 1 9/9
- Lig pSB1C3.N-CPP 2 9/9
- Lig pSB1C3.N-CPP * 6/9
- Cm 25 plates
Joint expression of SOD and yCCS from pEX
Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by Ahl, Lindberg and Tibell (2004), we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.
Extraction of RBS BioBrick (BBa_B0030)
Extracted BBa_B0030 (RBS 30), carried on pSB1A2, from iGEM plate 1, well 1H. Transformed into Top10.
- Quick transformation
- 1 μl DNA
- Amp 100
Cloning of His⋅SOD into pMA
Sequencing results
- pMA.his.SOD_premix (fasta)
Correct sequence verified by Blastn (results). Three silent mutations in the His-tag, as has been previously observed.
Mimmi
SOD.his / his.SOD / yCCS
Plasmid prep.
- Follow E.Z.N.A protocol
- Wash 2x with DNA wash buffer
- Eluate in 50µl sH2O