Team:SUSTC-Shenzhen/Notebook/Biobricks Characterization

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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 K916 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 restriction 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 (10.1 II)

Ligation (T)

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.

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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