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Team:SUSTC-Shenzhen/Notebook/Biobricks Characterization
From 2014.igem.org
Notebook
Biobricks Characterization
Contents |
Scheme
At first, we want to characterize plasmid assembled by 3 promoters, 3 RBSs, and 4 chromoprotein (36). Because time limits, we choose 2 promoter, 2 RBSs and 4 chromoprotein (16). In carrying out experiments, we cannot easily differ new constructed plasmid with BBa_E1010 with the self-assembly one. We abandoned BBa_E1010 and do experiments on other chromoproteins.
Results
We successfully constructed 8 parts, and they all are characterized. And 6 parts were sent to Registry of Standard Biological Parts. See them | HERE.
Procedures
- Amplification of Biobricks
- Add RBS
- Add promoter
- Add terminator
Plasmid Construction
ALL ABBREVIATIONS USED:
| Parts name | Abbreviations | Parts name | Abbreviations |
|---|---|---|---|
| BBa_J23100 | J00 | BBa_E1010 | E10 |
| BBa_J23106 | J06 | BBa_K592009 | K09 |
| BBa_B0031 | B31 | BBa_K592011 | K11 |
| BBa_B0034 | B34 | BBa_K1033916 | K916 |
| BBa_B0015 | B15 | BBa_I20260 | None |
| BBa_J04450 | None |
9.29
After RBS added, all seven plasmid were cut and ligated with two promoter, J23101 and J23106 respectively.
Enzyme digestion
For plan A
| J00 | J06 | B31E10 | B31K916 | B31K09 | B34E10 | B34K916 | B34 K09 | B34K11 | |
|---|---|---|---|---|---|---|---|---|---|
| EcoRI-HF(μL) | 1 | ||||||||
| XbaI(μL) | 1 | ||||||||
| PstI | 1 | ||||||||
| EcoRV-HF | 1 | ||||||||
| NcoI | 1 | 1 | |||||||
| Linearized
Backbone(μL) | 1 | ||||||||
| DNA(μL) | 3 | 4 | 8 | 7 | 4 | 5 | 5 | 5 | 5 |
| 10X NEB Buffer 2.1(μL) | 5 | ||||||||
| ddH2O (μL) | 39 | 38 | 34 | 35 | 38 | 37 | 37 | 37 | 37 |
| Total(μL) | 40 | ||||||||
For plan B
| J00 | J06 | B31
E10 | B31
K916 | B31
K09 | B34
E10 | B34
K916 | B34 K09 | B34
K11 | |
|---|---|---|---|---|---|---|---|---|---|
| EcoRI-HF(μL) | 1 | ||||||||
| XbaI(μL) | 1 | ||||||||
| PstI | 1 | ||||||||
| NcoI | 1 | 1 | 1 | ||||||
| Linearized
Backbone(μL) | 1 | ||||||||
| DNA(μL) | 3 | 4 | 8 | 7 | 4 | 5 | 5 | 5 | 5 |
| 10X NEB
Buffer 2.1(μL) | 5 | ||||||||
| ddH2O (μL) | 39 | 38 | 34 | 35 | 38 | 37 | 37 | 37 | 37 |
| Total(μL) | 40 | ||||||||
II.DNA Purification
III.Ligation
To complete construction quickly, we use 3A assembly to achieve plasmid with resistant to chloramphenicol (A) and standard assembly with resistant to Ampicillin (B).
Third step ligation - plan A(3A assembly)
| B331E10 | B31K916 | B31K09 | B34E10 | B34K916 | B34K09 | B34K11 | |
|---|---|---|---|---|---|---|---|
| DNA(50μg) | 4.0μL | 4.0μL | 2.0μL | 4.0μL | 2.0μL | 2.0μL | 4.0μL |
| 10x T4 Ligase
Buffer | 2.0μL | ||||||
| T4 Ligase | 1.0μL | ||||||
| ddH2O | 7.0μL | 7.0μL | 9.0μL | 7.0μL | 9.0μL | 9.0μL | 7.0μL |
| J23100(50μg) | 2.0μL | ||||||
| J23106(50μg) | 2.0μL | ||||||
| Backbone(50μg) | 2.0μL | ||||||
| Total | 10μL | ||||||
Ligation
To complete construction quickly, we use 3A assembly to achieve plasmid with resistant to chloramphenicol (A) and standard assembly with resistant to Ampicillin (B).
Ligation: In PCR system, 16 to ligate, 65℃ to inactive, and store at 4℃.
Transformation
- Place 7 EP tubes of 100μL DH5α competent cells on ice from -80℃ to melt.
- Transfer 50μL competent cells to 7 new sterilized EP tubes from each tubes in 1.
- Add 10μL of DNA to one EP tube with competent cells respectively.
- Put all EP tubes on ice for 30mins.
- Incubate in water at 42℃ for 90 seconds, then immediately on ice for 2 minutes.
- Add 200μL SOC broth, then put in a shaking incubator for 40 minutes at 37℃ , 220rpm.
- Centrifuge at 4500rpm for 2minutes, dispose 200μL supernatant.
- Resuspend competent cells and spread plates.
Incubate at 37
Characterization
References
- [http://www.tiangen.com/en/?productShow/t1/4/id/32.html |TIANprep Mini Plasmid Kit]
- [http://www.tiangen.com/en/?productShow/t1/4/id/41.html |TIANprep Midi Purification Kit]
- |NEB Biobricks® Assembly Kit
