Team:Peking/Notebook/HPan

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Heng Pan's Notes

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

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July

[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)

August

[TOP]

8.1

Miniprep:PbrR(PSB1K3)

Amplification of RBS+PbrR

8.2

Miniprep:RBS+PbrR

Digestion and identification by Electrophoresis

RBS+PbrR XbaI and PstI

Positive Transformation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator again into strain BL21 (DE3)

Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)

8.3

Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

8.4

Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator, but there is no colony of Pc promoter(1-18C)+RBS+PbrR

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

N_B/B_X/B_SD/SD_B/PET21A/RBS+PbrR

Ligation of Pc promoter(1-18C) and RBS+PbrR again

Induce strain BL21(DE3)(consist of plasmid T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator) by iptg again to gain the expression of GFP, but there were still no obvious difference between strain induced and uninduced.

8.5

Bacterial colonies PCR

Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

Transformation of Pc promoter(1-18C)+RBS+PbrR again.

Digestion and identification by Electrophoresis again

N_B/B_X/B_SD/SD_B/PET21A

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

Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

Ligation of N_B, B_X(insert) and PET21A, B_SD, SD_B,PSB1K3

Transformation of PET21A-NX and PSB3K3-BSD

Amplification of Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again

8.6

Amplification of Pc promoter(1-18C)+RBS+PbrR again

Bacterial colonies PCR again, but the result had been failed.

Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

Miniprep: Pc promoter(1-18C)+RBS+PbrR

Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again

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

Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

8.7

PbrR PCR, 50 uL system with Easy Pfu DNA Polymerase for the second time

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

PbrR XbaI and PstI

RBS(B0034) SpeI and PstI

Ligation of PbrR(insert) and RBS(B0034)(vector)

8.8

Bacterial colonies PCR again, but the result had been failed.

PbrR+RBS

Amplification of RBS(B0034)

8.9

Digestion and identification by Electrophoresis

PbrR(PSB1K3) XbaI and PstI

RBS(B0034) SpeI and PstI

Ligation of PbrR(insert) and RBS(vector) for the second time

Transformation of RBS+PbrR again

8.10

Digestion and identification by Electrophoresis

PbrR(PCR product) XbaI and PstI

Ligation of PbrR(insert) and PSB1K3(vector)

Transformation fo PbrR(PSB1K3)

8.11

Bacterial colonies PCR , PbrR(PSB1K3)

PbrD/PbrT PCR, 50 uL system with Easy Pfu DNA Polymerase

Indentification by Electrophoresis, but the PbrT had been failed.

PbrT PCR, 50 uL system with Easy Pfu DNA Polymerase again.

Digestion and identification by Electrophoresis: PbrD and PSB1C3(backbone)

8.12

Digestion and identification by Electrophoresis: PbrR(PSB1K3)/PbrT

Positive transformation of RBS(B0034)

Ligation of PbrD(insert) and PSB1C3(vector), PbrT(insert) and PSB1C3(vector)

Miniprep: RBS(B0034)

Digestion and identification by Electrophoresis: RBS(B0034)

8.13

Transformation of PbrD(PSB1C3), PbrT(PSB1C3), but there were no colony for them.

Ligation of PbrD/PbrT/PbrR(insert) and RBS(B0034)(vector)

8.14

PbrD/PbrT/PbrR PCR, 50 uL system with Easy Taq DNA Polymerase again, but the result of PbrT had been failed.

Ligation of PbrD(insert) and PSB1C3(vector), PbrT(insert) and PSB1C3(vector) again

8.15

Amplification of PbrT+RBS and PbrD+RBS

Digestion and identification by Electrophoresis: PbrD and PbrR again

8.16

Miniprep: RBS+PbrT and RBS+PbrD

Digestion and identification by Electrophoresis: RBS+PbrT and RBS+PbrD

8.17

Miniprep: RBS+PbrT, RBS+PbrR and RBS+PbrD

Digestion and identification by Electrophoresis: RBS+PbrT, RBS+PbrR and RBS+PbrD

Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector)

Transformation of RBS+PbrT+Terminator and T7 promoter+RBS+PbrD

8.18

Digestion and identification by Electrophoresis:RBS+PbrR

8.19

Miniprep: RBS+PbrT+Terminator and T7 promoter+RBS+PbrD

Digestion and identification by Electrophoresis: RBS+PbrT+Terminator and T7 promoter+RBS+PbrD

8.20

Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector)

8.24

Amplification of RBS+PbrR

Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) again

8.25

Miniprep: RBS+pbrR

Transformation of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator

Digestion and identification by Electrophoresis: RBS+PbrR

8.26

Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector)

8.27

Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

8.28

Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator

8.30

Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 promoter+RBS+Terminator+RBS+PbrT+Terminator

8.31

Ligation of MerP/PpbrA(insert) and CrtebI(vector)

September

[TOP]

9.1

Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into bacterium which consist of plasmid PpbrA+RBS+GFP, but there is no expression of GFP

9.2

The sequencing results showed that the sequence of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect

PbrR PCR, 50 uL system with Easy Taq DNA Polymerase for the fourth time

Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third time

9.3

Retrieve the PCR product and identification by Electrophoresis

Transformation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third

Ligation of PbrR(insert) and RBS(vector)

9.4

Amplification of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator

Transformation of RBS+PbrR

9.5

Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator

Amplification of RBS+PbrR

Digestion and identification by Electrophoresis: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator

9.6

The sequencing results showed that the sequence of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect again

Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector)

Transformation of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD

9.7

Amplification of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD

9.8

Miniprep: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD

Digestion and identification by Electrophoresis: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD

9.10

Ligation of T7promoter+RBS+PbrD(insert) and RBS+PbrT+Terminator(vector).

Transformation of T7promoter+RBS+PbrD+RBS+PbrT+Terminator

9.11

Amplification of T7promoter+RBS+PbrD+RBS+PbrT+Terminator

Digestion and identification by Electrophoresis: RBS+PbrR

Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector)

9.12

Miniprep: T7promoter+RBS+PbrD+RBS+PbrT+Terminator

Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

Digestion and identification by Electrophoresis: T7promoter+RBS+PbrD+RBS+PbrT+Terminator

9.13

The sequencing result of T7promoter+RBS+PbrD+RBS+PbrT+Terminator had failed again.

Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

9.14

Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR

9.15

Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into PpbrA+GFP generator to detect the expression level of GFP. But there were no expression of GFP.

9.16

The sequencing result showed than the sequence of PbrR was not intact.

9.17-9.21

Repeat the similar procedures to construct Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again.

9.22

Detect the expression level of GFP for the second time, but there were still no expression.

9.23

The sequencing result showed than the sequence of PbrR was not intact, which was similar to the sequence we had got before and Zhang Haoqian found that there was a PstI digest site in the sequence of PbrR

9.24

Transformation of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator into stain Trans 5a to detect the expression level of GFP

9.25

Ligation of Pc promoter+T7 polymerase(insert) and PSB3C5(backbone)

9.26

Miniprep: Pc promoter+T7 polymerase(PSB3C5)

Digestion and identification by Electrophoresis: Pc promoter+T7 polymerase(PSB3C5)

9.27

Transformation Pc promoter+T7 polymerase into bacterium with T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator to detect the expression level of GFP

9.28

Amplification of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator and Pc promoter+T7 polymerase.

9.29

Detect the expression level of GFP

@@ Line 492: / Line 492: @@
 |}
 [[https://2010.igem.org/Team:Peking/Notebook/HPan TOP]]
+===9.1===
+Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into bacterium which consist of plasmid
+PpbrA+RBS+GFP, but there is no expression of GFP
+===9.2===
+The sequencing results showed that the sequence of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7
+promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect
+PbrR PCR, 50 uL system with Easy Taq DNA Polymerase for the fourth time
+Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third time
+===9.3===
+Retrieve the PCR product and identification by Electrophoresis
+Transformation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third
+Ligation of PbrR(insert) and RBS(vector)
+===9.4===
+Amplification of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
+Transformation of RBS+PbrR
+===9.5===
+Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
+Amplification of RBS+PbrR
+Digestion and identification by Electrophoresis: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
+===9.6===
+The sequencing results showed that the sequence of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect again
+Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector)
+Transformation of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
+===9.7===
+Amplification of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
+===9.8===
+Miniprep: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
+Digestion and identification by Electrophoresis: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
+===9.10===
+Ligation of T7promoter+RBS+PbrD(insert) and RBS+PbrT+Terminator(vector).
+Transformation of T7promoter+RBS+PbrD+RBS+PbrT+Terminator
+===9.11===
+Amplification of T7promoter+RBS+PbrD+RBS+PbrT+Terminator
+Digestion and identification by Electrophoresis: RBS+PbrR
+Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector)
+===9.12===
+Miniprep: T7promoter+RBS+PbrD+RBS+PbrT+Terminator
+Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+Digestion and identification by Electrophoresis: T7promoter+RBS+PbrD+RBS+PbrT+Terminator
+===9.13===
+The sequencing result of T7promoter+RBS+PbrD+RBS+PbrT+Terminator had failed again.
+Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+===9.14===
+Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+===9.15===
+Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into PpbrA+GFP generator to detect the expression
+level of GFP. But there were no expression of GFP.
+===9.16===
+The sequencing result showed than the sequence of PbrR was not intact.
+===9.17-9.21===
+Repeat the similar procedures to construct Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again.
+===9.22===
+Detect the expression level of GFP for the second time, but there were still no expression.
+===9.23===
+The sequencing result showed than the sequence of PbrR was not intact, which was similar to the sequence we had got before and Zhang Haoqian found that there was a PstI digest site in the sequence of PbrR
+===9.24===
+Transformation of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator into stain Trans 5a to detect the expression level of GFP
+===9.25===
+Ligation of Pc promoter+T7 polymerase(insert) and PSB3C5(backbone)
+===9.26===
+Miniprep: Pc promoter+T7 polymerase(PSB3C5)
+Digestion and identification by Electrophoresis: Pc promoter+T7 polymerase(PSB3C5)
+===9.27===
+Transformation Pc promoter+T7 polymerase into bacterium with T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS
+generator to detect the expression level of GFP
+===9.28===
+Amplification of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator and Pc promoter+T7 polymerase.
+===9.29===
+Detect the expression level of GFP

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Team:Peking/Notebook/HPan

From 2010.igem.org

Revision as of 04:31, 26 October 2010

Contents

July

7.3

7.4

7.5

7.6

7.7

7.8

7.9

7.10

7.11

7.12

7.13

7.14

7.15

7.16

7.17

7.25

7.26

7.27

7.28

7.29

7.30

7.31

August

8.1

8.2

8.3

8.4

8.5

8.6

8.7

8.8

8.9

8.10

8.11

8.12

8.13

8.14

8.15

8.16

8.17

8.18

8.19

8.20

8.24

8.25

8.26

8.27

8.28

8.30

8.31

September

9.1

9.2

9.3

9.4

9.5

9.6

9.7

9.8

9.10

9.11

9.12

9.13

9.14

9.15

9.16

9.17-9.21

9.22

9.23

9.24

9.25

9.26

9.27

9.28

9.29

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	-	-	1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

Mon	Tue	Wed	Thu	Fri	Sat	Sun
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30	31	-	-	-	-

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	-	-	-