Team:SUSTC-Shenzhen/Notebook/Biobricks Characterization

From 2014.igem.org

(Difference between revisions)
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='''Scheme'''=
='''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 [https://2014.igem.org/Team:SUSTC-Shenzhen/Parts | HERE].
 +
 +
{{SUSTC-Image|wiki/images/e/ef/SUSTC-Shenzhen_Characterization_Parts.png|Parts}}
=='''Procedures'''==
=='''Procedures'''==
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After RBS added, all seven plasmid were cut and ligated with two promoter, J23101 and J23106 respectively.
After RBS added, all seven plasmid were cut and ligated with two promoter, J23101 and J23106 respectively.
==='''Enzyme digestion'''===
==='''Enzyme digestion'''===
-
 
+
For plan A
 +
{| class=''table''
 +
!
 +
!J00
 +
!J06
 +
!B31E10
 +
!B31K916
 +
!B31K09
 +
!B34E10
 +
!B34K916
 +
!B34K09
 +
!B34K11
 +
|-
 +
|EcoRI-HF(μL)
 +
|colspan='9'|1.0
 +
|-
 +
|XbaI(μL)
 +
|colspan='9'|1.0
 +
|-
 +
|PstI
 +
|colspan='2'|
 +
|colspan='7'|1.0
 +
|-
 +
|NcoI
 +
|colspan='2'|1.0
 +
|colspan='6'|
 +
|1.0
 +
|-
 +
|Linearized backbone(μL)
 +
|colspan='9'|1.0
 +
|-
 +
|DNA(μL)
 +
|3.0
 +
|4.0
 +
|8.0
 +
|7.0
 +
|4.0
 +
|colspan='4'|5.0
 +
|-
 +
|10x NEB Buffer 2.1(μL)
 +
|5.0
 +
|-
 +
|ddH2O(μL)
 +
|39
 +
|38
 +
|34
 +
|35
 +
|38
 +
|colspan='4'|37
 +
|-
 +
|Total(μL)
 +
|colspan='9'|40
 +
|
 +
}
==='''Ligation'''===
==='''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).
To complete construction quickly, we use 3A assembly to achieve plasmid with resistant to chloramphenicol (A) and standard assembly with resistant to Ampicillin (B).

Revision as of 07:05, 17 October 2014

Team SUSTC-Shenzhen

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.

Parts

Procedures

  1. Amplification of Biobricks
  2. Add RBS
  3. Add promoter
  4. Add terminator

Plasmid Construction

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 B34K09 B34K11
EcoRI-HF(μL) 1.0
XbaI(μL) 1.0
PstI 1.0
NcoI 1.0 1.0
Linearized backbone(μL) 1.0
DNA(μL) 3.0 4.0 8.0 7.0 4.0 5.0
10x NEB Buffer 2.1(μL) 5.0
ddH2O(μL) 39 38 34 35 38 37
Total(μL) 40

}

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

  1. Place 7 EP tubes of 100μL DH5α competent cells on ice from -80℃ to melt.
  2. Transfer 50μL competent cells to 7 new sterilized EP tubes from each tubes in 1.
  3. Add 10μL of DNA to one EP tube with competent cells respectively.
  4. Put all EP tubes on ice for 30mins.
  5. Incubate in water at 42℃ for 90 seconds, then immediately on ice for 2 minutes.
  6. Add 200μL SOC broth, then put in a shaking incubator for 40 minutes at 37℃ , 220rpm.
  7. Centrifuge at 4500rpm for 2minutes, dispose 200μL supernatant.
  8. Resuspend competent cells and spread plates.

Incubate at 37

Characterization

References

  1. [http://www.tiangen.com/en/?productShow/t1/4/id/32.html |TIANprep Mini Plasmid Kit]
  2. [http://www.tiangen.com/en/?productShow/t1/4/id/41.html |TIANprep Midi Purification Kit]
  3. |NEB Biobricks® Assembly Kit


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Licensed under CC BY 4.0.

  • ©2014 SUSTC-Shenzhen
Retrieved from "http://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/Biobricks_Characterization"