Team:SUSTC-Shenzhen/Notebook/Biobricks Characterization

From 2014.igem.org

(Difference between revisions)
Line 24: Line 24:
='''Plasmid Construction'''=
='''Plasmid Construction'''=
 +
ALL ABBREVIATIONS USED:
 +
{| class="table"
 +
! 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'''==
-
9.29
+
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
For plan A
-
{| class=''table''
+
{| class="wikitable"
-
!
+
!  
-
!J00
+
! J00
-
!J06
+
! J06
-
!B31E10
+
! B31E10
-
!B31K916
+
! B31K916
-
!B31K09
+
! B31K09
-
!B34E10
+
! B34E10
-
!B34K916
+
! B34K916
-
!B34K09
+
! B34 K09
-
!B34K11
+
! B34K11
|-
|-
-
|EcoRI-HF(μL)
+
| EcoRI-HF(μL)
-
|colspan='9'|1.0
+
| colspan="9" | 1
|-
|-
-
|XbaI(μL)
+
| XbaI(μL)
-
|colspan='9'|1.0
+
| colspan="9" | 1
|-
|-
-
|PstI
+
| PstI
-
|colspan='2'|
+
| colspan="2" |  
-
|colspan='7'|1.0
+
| colspan="6" | 1
 +
|
|-
|-
-
|NcoI
+
| EcoRV-HF
-
|colspan='2'|1.0
+
| colspan="2" |  
-
|colspan='6'|
+
| colspan="6" | 1
-
|1.0
+
|  
|-
|-
-
|Linearized backbone(μL)
+
| NcoI
-
|colspan='9'|1.0
+
| colspan="2" | 1
 +
| colspan="6" |
 +
| 1
|-
|-
-
|DNA(μL)
+
| Linearized
-
|3.0
+
Backbone(μL)
-
|4.0
+
| colspan="9" | 1
-
|8.0
+
-
|7.0
+
-
|4.0
+
-
|colspan='4'|5.0
+
|-
|-
-
|10x NEB Buffer 2.1(μL)
+
| DNA(μL)
-
|5.0
+
| 3
 +
| 4
 +
| 8
 +
| 7
 +
| 4
 +
| 5
 +
| 5
 +
| 5
 +
| 5
|-
|-
-
|ddH2O(μL)
+
| 10X NEB Buffer 2.1(μL)
-
|39
+
| colspan="9" | 5
-
|38
+
-
|34
+
-
|35
+
-
|38
+
-
|colspan='4'|37
+
|-
|-
-
|Total(μL)
+
| ddH2O (μL)
-
|colspan='9'|40
+
| 39
-
|
+
| 38
-
}
+
| 34
 +
| 35
 +
| 38
 +
| 37
 +
| 37
 +
| 37
 +
| 37
 +
|-
 +
| Total(μL)
 +
| colspan="9" | 40
 +
|}
 +
 
 +
For plan B
 +
{| class="table"
 +
!
 +
! J00
 +
! J06
 +
! B31
 +
E10
 +
! B31
 +
K916
 +
! B31
 +
K09
 +
! B34
 +
E10
 +
! B34
 +
K916
 +
! B34 K09
 +
! B34
 +
K11
 +
|-
 +
| EcoRI-HF(μL)
 +
| colspan="9" | 1
 +
|-
 +
| XbaI(μL)
 +
| colspan="9" | 1
 +
|-
 +
| PstI
 +
| colspan="2" |
 +
| colspan="7" | 1
 +
|-
 +
| NcoI
 +
| colspan="2" | 1
 +
| colspan="6" | 1
 +
| 1
 +
|-
 +
| Linearized
 +
Backbone(μL)
 +
| colspan="9" | 1
 +
|-
 +
| DNA(μL)
 +
| 3
 +
| 4
 +
| 8
 +
| 7
 +
| 4
 +
| 5
 +
| 5
 +
| 5
 +
| 5
 +
|-
 +
| 10X NEB
 +
Buffer 2.1(μL)
 +
| colspan="9" | 5
 +
|-
 +
| ddH2O (μL)
 +
| 39
 +
| 38
 +
| 34
 +
| 35
 +
| 38
 +
| 37
 +
| 37
 +
| 37
 +
| 37
 +
|-
 +
| Total(μL)
 +
| colspan="9" | 40
 +
|}
 +
 
 +
II.DNA Purification<br>
 +
III.Ligation<br>
 +
To complete construction quickly, we use 3A assembly to achieve plasmid with resistant to chloramphenicol (A) and standard assembly with resistant to Ampicillin (B). <br>
 +
Third step ligation - plan A(3A assembly)
 +
{| class="table"
 +
!
 +
! 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
 +
| colspan="7" | 2.0μL
 +
|-
 +
| T4 Ligase
 +
| colspan="7" | 1.0μL
 +
|-
 +
| ddH2O
 +
| 7.0μL
 +
| 7.0μL
 +
| 9.0μL
 +
| 7.0μL
 +
| 9.0μL
 +
| 9.0μL
 +
| 7.0μL
 +
|-
 +
| J23100(50μg)
 +
| colspan="7" | 2.0μL
 +
|-
 +
| J23106(50μg)
 +
| colspan="7" | 2.0μL
 +
|-
 +
| Backbone(50μg)
 +
|colspan="7" | 2.0μL
 +
|-
 +
| Total
 +
|10μL
 +
|}
 +
 
 +
 
 +
 
 +
 
==='''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).
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Incubate at 37
Incubate at 37
='''Characterization'''=
='''Characterization'''=
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 

Revision as of 07:28, 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

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

  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


Maintained by the iGEM team SUSTC-Shenzhen.

Licensed under CC BY 4.0.

  • ©2014 SUSTC-Shenzhen

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