Team:Peking/Notebook/HPan

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[[https://2010.igem.org/Team:Peking/Notebook/HPan TOP]]
[[https://2010.igem.org/Team:Peking/Notebook/HPan TOP]]
 +
===7.3===
 +
Transformation of plasmid: Terminator(B0015) and T7 promoter(BBa_I719005)
 +
===7.4===
 +
PCR to standardize the MerT and MerP from plasmid NRI
 +
 +
Amplification of bacterium: Terminator(B0015) and T7 promoter(BBa_I719005)
 +
===7.5===
 +
Miniprep: Terminator(B0015) and T7 promoter(BBa_I719005)
 +
 +
Digestion and identification by Electrophoresis
 +
 +
Terminator(B0015)        EcoRI and XbaII
 +
 +
T7 promoter(BBa_I719005)  SpeI and PstI
 +
 +
phiR73 delta(BBa_I746352)  EcoRI and SpeI
 +
 +
PO promoter(BBa_I746361)  EcoRI and XbaI
 +
===7.6===
 +
Ligation of phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
 +
 +
Transformation of ligation mixture of phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
 +
===7.7===
 +
Amplification of bacterium: phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
 +
 +
Miniprep: phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
 +
===7.8===
 +
Digestion and identification by Electrophoresis, but the result had been failed.
 +
 +
phiR73 delta+Terminator  XbaI and PstI
 +
 +
RBS(B0032)            SpeI and PstI
 +
 +
Digestion and identification by Electrophoresis again
 +
===7.9===
 +
Ligation of phiR73 delta+Terminator(insert) and RBS(B0032)(vector)
 +
 +
Transformation of ligation mixture of phiR73 delta+Terminator(insert) and RBS(B0032)(vector)
 +
 +
Digestion and identification by Electrophoresis
 +
 +
Terminator(B0015)            XbaI and PstI
 +
 +
AraC                        XbaI and PstI
 +
 +
Miniprep: plasmid of backbone PSB1C3
 +
 +
===7.10===
 +
Amplification of RBS+phiR73 delta+Terminator
 +
===7.11===
 +
The amplification had failed and amplification again.
 +
===7.12===
 +
Miniprep: phiR73 RBS+delta+Terminator
 +
 +
Digestion and identification by Electrophoresis
 +
 +
RBS+phiR73 delta+Terminator    XbaI and PstI
 +
 +
===7.13===
 +
Ligation of RBS+phiR73 delta+terminator(insert) and T7 promoter((BBa_I719005))(vector)
 +
 +
Transformation of ligation mixture of RBS+phiR73 delta+Terminator and T7 promoter((BBa_I719005))(vector)
 +
===7.14===
 +
Amplification of T7 promoter+RBS+phiR73 delta+Terminator
 +
 +
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator
 +
 +
Digestion and identification by Electrophoresis
 +
 +
  T7 promoter+RBS+phiR73 delta+Terminator    EcoRI and SpeI
 +
===7.15===
 +
Ligation of T7 promoter+RBS+phiR73 delta+Terminator(insert) and PO promoter(BBa_I746361)(vector)
 +
 +
Transformation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
 +
===7.16===
 +
Amplification of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
 +
 +
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
 +
===7.17===
 +
Digestion and identification by Electrophoresis
 +
 +
T7 promoter+RBS+phiR73 delta+Terminator+PO promoter  EcoRI and SpeI
 +
===7.25===
 +
Digestion and identification by Electrophoresis
 +
 +
  GFP generator(E0840)  EcoRI and Xbal
 +
 +
Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter(insert) and GFP generato T7 promoter+RBS+phiR73
 +
delta+Terminator+PO promoter+GFP generator r(E0840)(vector)
 +
 +
Transformation of Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
 +
===7.26===
 +
Amplification of Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
 +
===7.27===
 +
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
 +
 +
Digestion and identification by Electrophoresis
 +
 +
T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
 +
===7.28===
 +
Positive transformation T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator into strain BL21a
 +
===7.29===
 +
PbrR PCR, 50 uL system with Easy Pfu DNA Polymerase
 +
 +
Induce strain BL21a(consist of plasmid T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator) by iptg to gain the expression of GFP, but there were no obvious difference between strain induced and uninduced.
 +
===7.30===
 +
Retrieve the PCR product and identification by Electrophoresis
 +
 +
Digestion:
 +
 +
  PbrR  EcoRI and PstI/XbaI and PstI
 +
 +
Ligation of PbrR(insert) and PSB1K3(vector)
 +
 +
Transformation of PbrR(PSB1K3)
 +
===7.31===
 +
Ligation of PbrR(insert) and RBS(B0034)
 +
 +
Transformation of RBS+PbrR
 +
 +
Amplification of PbrR(PSB1K3)

Revision as of 04:15, 26 October 2010





   Heng Pan's Notes
                                                                                                                                                goto his page
I have constructed a genetic circuit, which utilizes the T3 polymerase/ T3 promoter and phage activator PhiR73/PO pomoter pairs to achieve a time dlay and signal amplification after mercury is present.Besides, I have characterized PpbrA-PbrR pair to stress that the engineering strategy applied in PmerT-MerR pair is robust for MerR family TFs


download his notes

Contents

July

Mon Tue Wed Thu Fri Sat Sun
- - - - 1 2 1
1 1 1 1 1 1 1
1 1 1 1 1 1 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

[TOP]

7.3

Transformation of plasmid: Terminator(B0015) and T7 promoter(BBa_I719005)

7.4

PCR to standardize the MerT and MerP from plasmid NRI

Amplification of bacterium: Terminator(B0015) and T7 promoter(BBa_I719005)

7.5

Miniprep: Terminator(B0015) and T7 promoter(BBa_I719005)

Digestion and identification by Electrophoresis

Terminator(B0015) EcoRI and XbaII

T7 promoter(BBa_I719005) SpeI and PstI

phiR73 delta(BBa_I746352) EcoRI and SpeI

PO promoter(BBa_I746361) EcoRI and XbaI

7.6

Ligation of phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)

Transformation of ligation mixture of phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)

7.7

Amplification of bacterium: phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)

Miniprep: phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)

7.8

Digestion and identification by Electrophoresis, but the result had been failed.

phiR73 delta+Terminator XbaI and PstI

RBS(B0032) SpeI and PstI

Digestion and identification by Electrophoresis again

7.9

Ligation of phiR73 delta+Terminator(insert) and RBS(B0032)(vector)

Transformation of ligation mixture of phiR73 delta+Terminator(insert) and RBS(B0032)(vector)

Digestion and identification by Electrophoresis

Terminator(B0015) XbaI and PstI

AraC XbaI and PstI

Miniprep: plasmid of backbone PSB1C3

7.10

Amplification of RBS+phiR73 delta+Terminator

7.11

The amplification had failed and amplification again.

7.12

Miniprep: phiR73 RBS+delta+Terminator

Digestion and identification by Electrophoresis 

RBS+phiR73 delta+Terminator     XbaI and PstI

7.13

Ligation of RBS+phiR73 delta+terminator(insert) and T7 promoter((BBa_I719005))(vector)

Transformation of ligation mixture of RBS+phiR73 delta+Terminator and T7 promoter((BBa_I719005))(vector)

7.14

Amplification of T7 promoter+RBS+phiR73 delta+Terminator

Miniprep: T7 promoter+RBS+phiR73 delta+Terminator

Digestion and identification by Electrophoresis 

  T7 promoter+RBS+phiR73 delta+Terminator     EcoRI and SpeI

7.15

Ligation of T7 promoter+RBS+phiR73 delta+Terminator(insert) and PO promoter(BBa_I746361)(vector)

Transformation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter

7.16

Amplification of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter

Miniprep: T7 promoter+RBS+phiR73 delta+Terminator+PO promoter

7.17

Digestion and identification by Electrophoresis

T7 promoter+RBS+phiR73 delta+Terminator+PO promoter EcoRI and SpeI

7.25

Digestion and identification by Electrophoresis 

  GFP generator(E0840)  EcoRI and Xbal

Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter(insert) and GFP generato T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator r(E0840)(vector)

Transformation of Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator

7.26

Amplification of Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator

7.27

Miniprep: T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator

Digestion and identification by Electrophoresis

T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator

7.28

Positive transformation T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator into strain BL21a

7.29

PbrR PCR, 50 uL system with Easy Pfu DNA Polymerase

Induce strain BL21a(consist of plasmid T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator) by iptg to gain the expression of GFP, but there were no obvious difference between strain induced and uninduced.

7.30

Retrieve the PCR product and identification by Electrophoresis

Digestion: 

  PbrR   EcoRI and PstI/XbaI and PstI

Ligation of PbrR(insert) and PSB1K3(vector)

Transformation of PbrR(PSB1K3)

7.31

Ligation of PbrR(insert) and RBS(B0034)

Transformation of RBS+PbrR

Amplification of PbrR(PSB1K3)

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