Team:SUSTC-Shenzhen/Notebook/Biobricks Characterization

From 2014.igem.org

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Digest overnight.
Digest overnight.
 +
=='''10.2'''==
=='''10.2'''==
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Isolate three colonies from plates of J00 B31 K916, J06 B31 K916, J06 B34 K916, J06 B34 K09, J06 B34 K11 to tubes with 3mL LB broth respectively.
Isolate three colonies from plates of J00 B31 K916, J06 B31 K916, J06 B34 K916, J06 B34 K09, J06 B34 K11 to tubes with 3mL LB broth respectively.
 +
=='''10.3'''==
=='''10.3'''==
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B31K11 1~3 were cut with E, P, E&P respectively totally 9 reactions.<br>
B31K11 1~3 were cut with E, P, E&P respectively totally 9 reactions.<br>
Plates (10.2) grow single colonies: J00 B34 E10, J00 B34 K09, J00 B34 K09 isolate single colonies preparing for extraction.
Plates (10.2) grow single colonies: J00 B34 E10, J00 B34 K09, J00 B34 K09 isolate single colonies preparing for extraction.
 +
=='''10.5'''==
=='''10.5'''==
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| style="text-align: center;" | 10
| style="text-align: center;" | 10
|}
|}
 +
=='''10.6'''==
=='''10.6'''==
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| colspan="6" style="text-align: center;" | 20
| colspan="6" style="text-align: center;" | 20
|}
|}
-
 
Transformaion<br>
Transformaion<br>
 +
 +
=='''10.8'''==
 +
 +
==='''Plasmid extraction'''===
 +
J00 B34 K11<br>
 +
 +
Isolate three single colonies from plates of J06 B34 K916, J00 B34 K09, J06 B34 K11,J06 B34 K11, J06 B31 K916 (10.6 II).<br>
 +
 +
==='''Ligation'''===
 +
{| class=''table"
 +
! style="text-align: center;" |
 +
! style="text-align: center;" | J00 B34 K916
 +
! style="text-align: center;" | J06 B31 K09
 +
! style="text-align: center;" | J00 B31 K09
 +
! style="text-align: center;" | J00 B31 K11
 +
! style="text-align: center;" | J06 B31 K11
 +
|-
 +
| style="text-align: center;" | J23100(μL)
 +
| style="text-align: center;" | 2
 +
| style="text-align: center;" |
 +
| style="text-align: center;" | 2
 +
| style="text-align: center;" | 2
 +
| style="text-align: center;" |
 +
|-
 +
| style="text-align: center;" | J23106(μL)
 +
| style="text-align: center;" |
 +
| style="text-align: center;" | 2
 +
| style="text-align: center;" |
 +
| style="text-align: center;" |
 +
| style="text-align: center;" | 2
 +
|-
 +
| style="text-align: center;" | DNA(μL)
 +
| colspan="5" style="text-align: center;" | 2
 +
|-
 +
| style="text-align: center;" | T4 Ligase(μL)
 +
| colspan="5" style="text-align: center;" | 0.4
 +
|-
 +
| style="text-align: center;" | T4 Ligase buffer(μL)
 +
| colspan="5" style="text-align: center;" | 2
 +
|-
 +
| style="text-align: center;" | ddH2O
 +
| colspan="5" style="text-align: center;" | 13.6
 +
|-
 +
| style="text-align: center;" | Total(μL)
 +
| colspan="5" style="text-align: center;" | 20
 +
|}
 +
 +
Transformation with DH5α
 +
 +
 +
=='''10.9'''==
 +
 +
I.Bacteria with 3A assembly plasmid grow so much slowly.<br>
 +
 +
II. Transformation (10.8) was done with DH5α, we have to do again with BL21.<br>
 +
 +
III.Broth culture, plasmid extraction and storage of complete plasmid J00 B34 K09+T, J00 B34K916+T, J06 B34 K09+T, J06 B34 K916+T and J06 B31 K916+T. And extra broth were sent to be sequenced. Unfortunately, EP tubes of J00 B34 K916+T and J06 B31 K916+T were mixed. We recovered broth culture and reinoculated 200μL to 3mL LB Broth, and do extraction again.<br>
 +
 +
V.Plasmid extraction of J06 B31 K916 (without terminator).<br>
 +
 +
==='''Ligation'''===
 +
{| class="table"
 +
!
 +
! J00 B34 K916
 +
! J00 B31 K09
 +
! J00 B31 K11
 +
! J06 B31 K09
 +
! J06 B31 K11
 +
! J06 B34 K11+T
 +
|-
 +
| DNA(μL)
 +
| style="text-align: center;" | 3
 +
| style="text-align: center;" | 3
 +
| style="text-align: center;" | 3.5
 +
| style="text-align: center;" | 3
 +
| style="text-align: center;" | 3.5
 +
| style="text-align: center;" | 3
 +
|-
 +
| J23100(μL)
 +
| style="text-align: center;" | 1.5
 +
| style="text-align: center;" | 1.5
 +
| style="text-align: center;" | 1.5
 +
| style="text-align: center;" |
 +
| style="text-align: center;" |
 +
| style="text-align: center;" |
 +
|-
 +
| J23106(μL)
 +
| colspan="3" style="text-align: center;" |
 +
| style="text-align: center;" | 1.5
 +
| style="text-align: center;" | 2.5
 +
| style="text-align: center;" |
 +
|-
 +
| B0015(μL)
 +
| colspan="5" style="text-align: center;" |
 +
| style="text-align: center;" | 2.5
 +
|-
 +
| T4 Ligase buffer(μL)
 +
| colspan="6" style="text-align: center;" | 2
 +
|-
 +
| T4 Ligase(μL)
 +
| colspan="6" style="text-align: center;" | 1
 +
|-
 +
| ddH2O
 +
| style="text-align: center;" | 12.5
 +
| style="text-align: center;" | 12.5
 +
| style="text-align: center;" | 12
 +
| style="text-align: center;" | 12.5
 +
| style="text-align: center;" | 12
 +
| style="text-align: center;" | 11.5
 +
|-
 +
| Total(μL)
 +
| colspan="6" style="text-align: center;" | 20
 +
|}
 +
 +
Transformation：First five with BL21, J06 B34 K11+T with DH5α.<br>
 +
 +
VI.Because plasmid constructed with BBa_E1010 is difficult to be differed with J04450, we decided to abandon construction those with BBa_E1010.
 +
 +
 +
 +
=='''10.10'''==
 +
 +
==='''Enzyme digestion'''===
 +
{| class="table"
 +
!
 +
! J00 B31 K916
 +
! B0015
 +
|-
 +
| style="text-align: center;" | DNA(μL)
 +
| style="text-align: center;" | 22
 +
| style="text-align: center;" | 22
 +
|-
 +
| style="text-align: center;" | EcoRI-HF(μL)
 +
| style="text-align: center;" | 2
 +
| style="text-align: center;" | 2
 +
|-
 +
| style="text-align: center;" | SpeI(μL)
 +
| style="text-align: center;" | 2
 +
| style="text-align: center;" |
 +
|-
 +
| style="text-align: center;" | XbaI(μL)
 +
| style="text-align: center;" |
 +
| style="text-align: center;" | 2
 +
|-
 +
| style="text-align: center;" | 10x NEB buffer 2.1(μL)
 +
| style="text-align: center;" | 5
 +
| style="text-align: center;" | 5
 +
|-
 +
| style="text-align: center;" | ddH2O
 +
| style="text-align: center;" | 19
 +
| style="text-align: center;" | 19
 +
|-
 +
| style="text-align: center;" | Total(μL)
 +
| style="text-align: center;" | 50
 +
| style="text-align: center;" |
 +
|}
 +
 +
Transformation
 +
 +
 +
=='''10.11'''==
 +
 +
I. Gel electeophoresis<br>
 +
II. Gel extraction<br>
 +
==='''Ligation'''===
 +
{| class="table"
 +
! style="text-align: center;" |
 +
! style="text-align: center;" | J00 B31 K916
 +
|-
 +
| style="text-align: center;" | BOO15(Extraction)
 +
| style="text-align: center;" | 1.0
 +
|-
 +
| style="text-align: center;" | DNA
 +
| style="text-align: center;" | 2
 +
|-
 +
| style="text-align: center;" | T4 Ligase
 +
| style="text-align: center;" | 0.5
 +
|-
 +
| style="text-align: center;" | T4 Ligase
 +
buffer
 +
| style="text-align: center;" | 2
 +
|-
 +
| style="text-align: center;" | ddH2O
 +
| style="text-align: center;" | 13.5
 +
|-
 +
| style="text-align: center;" | Total
 +
| style="text-align: center;" | 20
 +
|}
 +
 +
Transformation
 +
 +
 +
 +
=='''10.12'''==
 +
I. Plates of (Ampicillin) J06 B31 K916+T, J00 B31 K11+T, J00 B31 K11+T and J00 B31 K09+T grew colonies and single colonies were isolated and broth.
 +
 +
II. Plates of J06 B31 K11, J06 B31 K09, J00 B31 K916 and B0015 grew no colonies.
 +
 +
III. Plates of J06 B34 K11+T (10.9 VI) grew single green colonies one of which was isolated and transfer to broth, and plasmid was extracted at night.
 +
 +
IV. Ligations for J06 B31 K09 and J06 B31 K11 were redid.
 +
==='''Ligation'''===
 +
{| class="table"
 +
! style="text-align: center;" |
 +
! style="text-align: center;" | J06 B31 K09
 +
! style="text-align: center;" | J06 B31 K11
 +
|-
 +
| style="text-align: center;" | J23106(Backbone)
 +
| colspan="2" style="text-align: center;" | 1.0μL
 +
|-
 +
| style="text-align: center;" | DNA(μL)
 +
| colspan="2" style="text-align: center;" | 2.0μL
 +
|-
 +
| style="text-align: center;" | T4 Ligase(μL)
 +
| style="text-align: center;" | 1.5μL
 +
| style="text-align: center;" | 3.0
 +
|-
 +
| style="text-align: center;" | T4 Ligase Buffer(μL)
 +
| style="text-align: center;" | 2μL
 +
| style="text-align: center;" | 4.0
 +
|-
 +
| style="text-align: center;" | ddH2O
 +
| style="text-align: center;" | 13.5
 +
| style="text-align: center;" | 10
 +
|-
 +
| style="text-align: center;" | Total(μL)
 +
| colspan="2" style="text-align: center;" | 20
 +
|}
 +
 +
 +
=='''10.13'''==
 +
==='''Plasmid extraction'''===
 +
J06 B34 K11+T.
 +
 +
 +
 +
=='''10.14'''==
 +
I.Transformation of J00 B34 K11+T
 +
II.Isolate three single colonies from each plates of J00 B31 K916+T, J00 B34 K11+T, J06 B34 K11+T.
 +
III. Plasmid extraction
 +
IV. Plates of J00 B31 K09 and J00 B31 K11 is not distinct, we decided to abandon these constructions.

Revision as of 11:31, 17 October 2014

Team SUSTC-Shenzhen

Notebook

Biobricks Characterization

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

DNA Purification

Follow instructions in kit.

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

Third step ligation -planB(Standard Assembly)

B31

E10

B31 K916 B31 K09 B34

E10

B34 K916 B34

K09

B34 K11
DNA(μL) 0.7 0.5 1.2 0.8 1.2 1.4 0.7
J23100(μL) 1.2
J23106(μL) 1.0
T4 Ligase(μL) 0.5
T4 Ligase buffer(μL) 1
ddH2O(μL) 6.6 6.8 6.8

7.0

6.1

6.3

6.5

6.7

6.1

6.3

5.9

6.1

6.6

6.8

Total(μL) 10

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℃.

9.30

I. 4 plates grew single colonies

J00 B34 E10
J00 B34 K91
J06 B31 K09
J06 B34 K09

Religation

B34 E10 B31 K09 B34 K09
DNA(50μg) 0.8μL
J23106 1.0μL 1.0μL
J23100 1.2μL
Buffer 1μL 1μL 1μL
Ligase 0.5μL 0.5μL 0.5μL
ddH2O 6.5μL 6.3μL 6.1μL
Total 20μL


Transformation

III. Isolate three colonies from plates of J06 B32 E10, J06 B31 K916, J06 B34 E10 and J00 B31 K09 to three tubes of 3ml LB broth respectively.

10.1

Plasmid extraction

J06 B31 K916, J00 B34 K916, J06 B34 K916, J06 B34 E10X2, J06 B31 E10 and J06 B31 K916. (Protocols following instructions in kit.) All these seven plasmid were digested with EcoRI, PstI, EcoRI-HF&PstI and go gel electrophoresis tests on 10.2. (Sample list has the same order in former text)

characterization

Enzyme Digestion I

J23100 J23106 B31E10 B31K916 B31K09 B34E10 B34K916 B34K09 B34K11
DNA(1μg 3.5μL 4.1μL 7.5μL 6.7μL 4.4μL 4.5μL 4.6μL 4.6μL 4.7μL
NEB Buffer 2.1 5μL 5μL 5.0μL 5.0μL 5.0μL 5.0μL 5.0μL 5.0μL 5.0μL
XbaI 0.6μL
SpeI 0.6μL 0.6μL
PstI 0.6μL
ddH2O 40.3μL 39.7μL 36.3μL 37.1μL 39.4μL 39.3μL 39.2μL 39.2μL 39.1μL
Total 50μL

Digest Overnight

Enzyme Digestion II

J06 B31 K916 J00 B34 K916 J06 B34 K916 J06 B34 E10-1 J06 B34 E10-2 J06 B31 E10 J06 B31 K916
DNA(μL) 2
Buffer(μL) 1
EcoRI-HF(μL) 0.3
PstI(μL) 0.3
ddH2O(μL) 6.4
Total(μL) 10

Digest overnight

Enzyme Digestion III

pSB1C3 RFC B31K09 B31 K916 B31 K09 B34 E10 B34 B34 K11 B34 K11 B31 J00 J06
XbaI(μL) 2
PstI(μL) 2
SpeI(μL) 2
DNA(μL) 12 15 13 9 9 10 10 10 10 11 7 9
10X NEB Buffer 2.1(μL) 10
ddH2O(μL) 74 71 73 77 77 76 76 76 76 75 79 77
Total 100

Digest overnight.


10.2

Gel electrophoresis

54μL reaction,9μL loading dye

Gel extraction

Ligation

J00 B31 E10 J00 B31 K09 J00 B34 E10 J00 B34 K09 J00 B34 K11 J06 B31 E10 J06 B31 K09 J06 B34 E10 B31 K11 J00 J06
Ligase(μL) 0.5
Buffer(μL) 1
Promoter

Backbone(μL)

1.3 0.9 1.3
DNA(μL) 1.2 1.5 1.6 1.6 1.6 1.2 1.5 1.6 1.4
ddH2O(μL) 6 5.7 5.6 5.6 5.6 6 5.7 5.6 6.2 7.2 7.2
Total(μL) 10

Isolate three colonies from plates of J00 B31 K916, J06 B31 K916, J06 B34 K916, J06 B34 K09, J06 B34 K11 to tubes with 3mL LB broth respectively.


10.3

Transformation(10.2)

Enzyme digestion

The five plasmid extracted on 10.1

J00 B31 K916 J06 B31 K916 J06 B34 K916 J06 B34 K09 J06 B34 K11 B0015
EcoRI-HF(μL) 2
SpeI(μL) 2
DNA(μL) 9 9 10 9 9 12
Buffer(μL) 10
ddH2O(μL) 77 77 76 77 77 74
Total(μL) 100


10.4

Gel electrophoresis 120μL (10.3)

Gel extraction

Ligation

Adding terminator and change backbone to pSB1C3.

J06 B31K916 J06B34 K916 J00B31 K916 J06 B34 K11 J06 B34 K09
B0015(Gel extraction) (μL) 1
DNA(μL) 2
T4 Ligase(μL) 0.5
T4 Ligase Buffer(μL) 1
ddH2O(μL) 5.5
Total(μL) 10

Transformation

Plasmid Extraction B31K11 1~3

Enzyme digestion

B31K11 1~3 were cut with E, P, E&P respectively totally 9 reactions.
Plates (10.2) grow single colonies: J00 B34 E10, J00 B34 K09, J00 B34 K09 isolate single colonies preparing for extraction.


10.5

Plasmid extraction=

J00 B34 E10, J00 B34 K09, J00 B34 K11

Gel electrophoresis

J23106 J23100 B31K11

Religation

Ligation products 10.2) grow slowly, which is out of expectation. We decided to redo the ligation.

J00 B31 E10 J00 B31 K09 J00 B34 E10 J00 B34 K11 J06 B31 E10 J06 B31 K09 J06 B34 E10
T4 Ligase(μL) 1
T4 Ligase Buffer(μL) 2
B0015(μL) 2.6
DNA(μL) 2.4 3 3.2 3.2 2.4 3.0 3.2
ddH2O(μL) 12 11.4 11.2 11.2 12 11.4 11.2
Total(μL) 20

Transformation

Enzyme digestion

(Preparing for ligation)

B31K11 J00 J06
XbaI(μL) 2
PstI(μL) 2
DNA(μL) 12 7 9
Buffer(μL) 10
ddH2O(μL) 74 79 77
Total(μL) 100

Gel electrophoresis'

Gel extraction

Ligation

B31K11
J00(μL) 2
J06(μL) 2
DNA(μL) 2
Ligase(μL) 0.5
Buffer(μL) 1
ddH2O(μL) 4.5
Total(μL) 10


10.6

(10.5) isolate 3 colonies from the plates.

Ligation

(Adding terminator and changing backbone)

J00 B34 K11 J00 B34 K09 J00 B31 K916 J06 B31 K916 J06 B34 K916 J06 B34 K11 J06 B34 K09
B0015(Gel extraction) (μL) 2
DNA(μL) 2
Ligase(μL) 0.5
Buffer(μL) 1
ddH2O(μL) 4.5
Total(μL) 10

Transformation

Enzyme digestion I

J00 B34 E10
Buffer (μL) 5
EcoRI-HF(μL) 1
SpeI(μL) 1
DNA(μL) 10
ddH2O(μL) 33
Total(μL) 50

Enzyme digestion II

J00 B34 E10 J00 B34 K09 J00 B34 K11 B0015-1 B0015-2 B0015-3
EcoRI-HF(μL) 2 1 2
SpeI(μL) 2 1
XbaI(μL) 2
Buffer(μL) 10 5 10
DNA(2) (μL) 13 14 18 5 5 10
ddH2O(μL) 73 72 68 39 39 74
Total(μL) 100 50 50 100

We suspected B0015 didn’t work well, and gel electrophoresis was done. B0015 was digested with XbaI, EcoRI-HF, XbaI&EcoRI-HF respectively. Gel electrophoresis


10.7

We checked Part Registry of B0015, and find it’s better to add B0015 behind the coding region instead of inserting protein, RBS and promoter into B0015.

3A assembly

B0015 I20260 J00 B31 K916 J00 B34 K09 J00 B34 K11 J06 B31 K916 J06 B34 K916 J06 B34 K09
EcoRI-HF(μL) 0.5 2
PstI(μL) 0.5 0.5 2
10x NEB Buffer 2.1(μL) 2 5
DNA(μL) 11 17 9 14 18 9 10 9
ddH2O(μL) 6 4 32 27 23 32 31 32
Total(μL) 20 50

Ligation I

J06 B31 K916 J06 B34 K916 J06 B34 K11 J06 B34 K09 J06 B31 K916 J00 B34 K09 J00 B34 K11
B0015(μL) 1
I20260(Backbone)(μL) 1
T4 Ligase(μL) 0.5
T4 Ligase buffer(μL) 2
ddH2O 13
Total(μL) 20


Transformation

Ligation II

J06 B31 E10 J06 B34 E10 J06 B34 K11 J00 B34 E10
DNA(μL) 10
T4 Ligase(μL) 0.8
T4 Ligase Buffer(μL) 3
ddH2O 14.2
Total(μL) 30

Ligation III

J06 B31 K916 J00 B31 K916 J06 B34 K916 J06 B34 K09 J00 B34 K11 J00 B34 K09
J04450(Backbone) (μL) 2.5
T4 Ligase(μL) 0.4
T4 Ligase buffer(μL) 014.6
DNA(μL) 2.5
ddH2O 14.6
Total(μL) 20

Transformaion


10.8

Plasmid extraction

J00 B34 K11

Isolate three single colonies from plates of J06 B34 K916, J00 B34 K09, J06 B34 K11,J06 B34 K11, J06 B31 K916 (10.6 II).

Ligation

J00 B34 K916 J06 B31 K09 J00 B31 K09 J00 B31 K11 J06 B31 K11
J23100(μL) 2 2 2
J23106(μL) 2 2
DNA(μL) 2
T4 Ligase(μL) 0.4
T4 Ligase buffer(μL) 2
ddH2O 13.6
Total(μL) 20

Transformation with DH5α


10.9

I.Bacteria with 3A assembly plasmid grow so much slowly.

II. Transformation (10.8) was done with DH5α, we have to do again with BL21.

III.Broth culture, plasmid extraction and storage of complete plasmid J00 B34 K09+T, J00 B34K916+T, J06 B34 K09+T, J06 B34 K916+T and J06 B31 K916+T. And extra broth were sent to be sequenced. Unfortunately, EP tubes of J00 B34 K916+T and J06 B31 K916+T were mixed. We recovered broth culture and reinoculated 200μL to 3mL LB Broth, and do extraction again.

V.Plasmid extraction of J06 B31 K916 (without terminator).

Ligation

J00 B34 K916 J00 B31 K09 J00 B31 K11 J06 B31 K09 J06 B31 K11 J06 B34 K11+T
DNA(μL) 3 3 3.5 3 3.5 3
J23100(μL) 1.5 1.5 1.5
J23106(μL) 1.5 2.5
B0015(μL) 2.5
T4 Ligase buffer(μL) 2
T4 Ligase(μL) 1
ddH2O 12.5 12.5 12 12.5 12 11.5
Total(μL) 20

Transformation：First five with BL21, J06 B34 K11+T with DH5α.

VI.Because plasmid constructed with BBa_E1010 is difficult to be differed with J04450, we decided to abandon construction those with BBa_E1010.


10.10

Enzyme digestion

J00 B31 K916 B0015
DNA(μL) 22 22
EcoRI-HF(μL) 2 2
SpeI(μL) 2
XbaI(μL) 2
10x NEB buffer 2.1(μL) 5 5
ddH2O 19 19
Total(μL) 50

Transformation


10.11

I. Gel electeophoresis
II. Gel extraction

Ligation

J00 B31 K916
BOO15(Extraction) 1.0
DNA 2
T4 Ligase 0.5
T4 Ligase

buffer

2
ddH2O 13.5
Total 20

Transformation


10.12

I. Plates of (Ampicillin) J06 B31 K916+T, J00 B31 K11+T, J00 B31 K11+T and J00 B31 K09+T grew colonies and single colonies were isolated and broth.

II. Plates of J06 B31 K11, J06 B31 K09, J00 B31 K916 and B0015 grew no colonies.

III. Plates of J06 B34 K11+T (10.9 VI) grew single green colonies one of which was isolated and transfer to broth, and plasmid was extracted at night.

IV. Ligations for J06 B31 K09 and J06 B31 K11 were redid.

Ligation

J06 B31 K09 J06 B31 K11
J23106(Backbone) 1.0μL
DNA(μL) 2.0μL
T4 Ligase(μL) 1.5μL 3.0
T4 Ligase Buffer(μL) 2μL 4.0
ddH2O 13.5 10
Total(μL) 20


10.13

Plasmid extraction

J06 B34 K11+T.


10.14

I.Transformation of J00 B34 K11+T II.Isolate three single colonies from each plates of J00 B31 K916+T, J00 B34 K11+T, J06 B34 K11+T. III. Plasmid extraction IV. Plates of J00 B31 K09 and J00 B31 K11 is not distinct, we decided to abandon these constructions.



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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