Team:Peking/Notebook/YHu

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Contents

• June, 2010

• July, 2010

• August, 2010

• September & October, 2010

June

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6.30

1. Dissolve primers: primers for mbp fragments (standardization): SD_ForA, SD_ForC, SD_Rev. primers for mbp fragments (commercialization): NdeI_A(For), NdeI_C(For), XhoI(Rev).

2. Miniprep: plasmids provided by Anne O Summers and I-120.

3. PCR reaction for mbp fragments using dissolved primers.

5x PHF buffer II 4uL

ddNTP Mixture 1.6uL

For 1uL

Rev 1uL

Template plasmid 0.5uL

phusionHF 0.2uL

ddH2O up to 20 uL

4. Electrophoresis PCR reaction system in 1% agarose gel.

5. Excise the gel slice and extract the mbp fragments.

July

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7.1

1. DNA double digestion of mbp fragments for 3 hours: NdeI/XhoI for commercialization and XbaI/PstI for standardization.

20uL reaction system:

Buffer4 2uL

NdeI 1.5uL

XhoI 1.5uL

mbp(com) 15uL

Buffer3 2uL

XbaI 1.5uL

PstI 1.5uL

mbp(SD) 15uL

2. DNA Purification.

7.17

1. PCR reaction for mbp fragments using dissolved primers.

Primers Reverse is the new ones with strepp tag.

10x easypfu buffer 2uL

ddNTP Mixture 2uL

For 1uL

Rev 1uL

Template plasmid 0.5uL

phusionHF 0.2uL

ddH2O up to 20 uL

2. Electrophoresis PCR reaction system in 1% agarose gel.

3. Excise the gel slice and extract the mbp fragments.

7.18

1. DNA double digestion of mbp fragments and merr plasmids.

2. ligation of insert DNA fragments into plasmid vector DNA for 1 hour

mbp(NdeI/XhoI) 6uL

pET-21a 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

mbp (Xbal/PstI) 6uL

B0034 (rbs) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

merr (Xbal/PstI) 6uL

B0034 (rbs) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

3. Transformation

7.19

1. Miniprep of rbs-merr

2. Digestion and identification by Electrophoresis

Buffer3 1uL

XbaI 0.75uL

PstI 0.75uL

rbs-merr 7.5uL

7.20

Miniprep of 12 colonies of mbp-commercialization.

No colonies formation of rbs-mbp.

7.21

1. DNA double digestion of colonies of mbp-pET-21a and rbs-merr.

10uL reaction system:

Buffer4 1uL

NdeI 0.75uL

XhoI 0.75uL

mbp-pET-21a 7.5uL

Buffer3 1uL

XbaI 0.75uL

PstI 0.75uL

rbs-mbp 7.5uL

2. Electrophoresis in 1% agarose gel.

Sequence mbp_ commercialization

(The result is confirmed to be incorrect.)

3. ligation of rbs-mbp-1 and 2 into plasmid vector T7 for 1 hour

rbs-mbp(Xbal/PstI) 6uL

T7 vector(SpeI/PstI) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

4. Transformation

7.22

No colonies formation of T7-rbs-merr. Do ligation and transformation again.

7.23

Picking colonies of T7-rbs-merr and shaking at 37℃ overnight.

7.24

Problems existed in our experiment: The PCR reaction product cannot be verified. New primers forward (3C and 3Plus) for mbp fragments are designed.

1. PCR reaction for mbp fragments using dissolved primers.

For 1uL

Rev 1uL

Easymix 10uL

template 0.2uL

ddH2O up to 20 uL

2. Miniprep of T7-rbs-merr

3. Digestion and identification by Electrophoresis

EcoRI Buffer 1uL

EcorRI 0.75uL

PstI 0.75uL

T7-rbs-merr 7.5uL

The band was not correct.

7.25

1. Electrophoresis PCR reaction product last day in 1% agarose gel.

2. Excise the gel slice and extract the mbp fragments.

3. DNA double digestion of mbp fragments for 3 hours: Xbal/XhoI for commercialization and XbaI/PstI for standardization.

20uL reaction system:

Buffer4 2uL

Xbal 1.5uL

XhoI 1.5uL

Product 15uL

Buffer3 2uL

XbaI 1.5uL

PstI 1.5uL

Product 15uL

2. DNA Purification.

3. ligation for 1 hour

mbp(XbaI/XhoI) 6uL

pET-21a 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

mbp (Xbal/PstI) 6uL

B0034 (rbs) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

7.26

Transformation of ligation mixure.

Name of plates were: 3C-com, 3Plus-com, 3C-SD, 3plus-SD

• com means commercialization and SD means standardization

7.27-7.28

No colonies formation of 3C-com and 3plus-com

1. Picking 18 colonies from 3C-SD and 3plus-SD(mbp_SD) and shaking at 37℃ for 10 hours.

2. Miniprep mbp_SD

3. Digestion and identification by Electrophoresis

EcoRI Buffer 1uL

EcorRI 0.75uL

PstI 0.75uL

mbp_SD 7.5uL

Sequence.

(The result is confirmed to be incorrect.)

7.30

Template of pASK-mbp is verified to have mutagenesis; therefore we decide to construct mbp by linking 2 merr fragments together.

1. PCR reaction for six merr fragments: N_B and B_X; SD(Xbal)_B and B_SD(PstI); SD(EcoRI)_B and B_SD(PstI).

10x buffer 5uL

ddNTP Mixture 5uL

For 1uL

Rev 1uL

Template plasmid 0.5uL

Polymerase 1uL

ddH2O up to 50 uL

2. Electrophoresis PCR reaction system in 1% agarose gel to identification.

3. DNA purification from reaction mixture.

4. DNA double digestion for 3 hours.

20uL reaction system:

N_B:

Buffer3 2uL

NdeI 1.5uL

BamHI 1.5uL

Product 15uL

B_X:

Buffer3 2uL

BamHI 1.5uL

XhoI 1.5uL

Product 15uL

SD(Xbal)_B:

Buffer3 2uL

XbaI 1.5uL

BamHI 1.5uL

Product 15uL

B_SD(PstI):

Buffer3 2uL

BamHI 1.5uL

PstI 1.5uL

Product 15uL

SD(EcoRI)_B:

EcoRI Buffer 2uL

EcorRI 1.5uL

BamHI 1.5uL

Product 15uL

5. DNA purification after the double digestion.

6. ligation of 2 merr fragments into the vector for 6 hours.

Insert1 3uL

Insert2 3uL

Vector 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

mbp-Com: Inserts: N_B and B_X; vector: pET-21a(NdeI/Xhol)

mbp-SD: Inserts: SD(Xbal)_B and B_SD(PstI), vector:B0034(SpeI/PstI)

mbp: SD(EcoRI)_B and B_SD(PstI), vector: pSB1K3 (EcoRI/PstI)

7. Transformation

7.31

No colonies formation of mbp-Com.

1. Picking colonies from mbp-SD and mbp, shaking at 37℃ for 10 hours.

2. Miniprep of mbp-SD and mbp

3. Miniprep of B0034

4. Double digestion for 3 hours.

Buffer2 2uL

SpeI 1.5uL

PstI 1.5uL

B0034(rbs) 5uL

ddH2O up to 20 uL

August

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8.1

1. mbp Digestion and identification by Electrophoresis

EcoRI Buffer 1uL

EcorRI 0.75uL

PstI 0.75uL

mbp 7.5uL

The band is not correct.

2. mbp-SD Double Digestion

Buffer3 2uL

XbaI 1.5uL

PstI 1.5uL

rbs-mbp_SD 5uL

ddH2O up to 20uL

3. Electrophoresis rbs-mbp-SD in 1% agarose gel.

4. Excise the gel slice and extract the rbs-mbp-SD fragments.

8.2

1. Ligation of rbs-mbp_SD plasmid vector T7 for 1 hour.

rbs-mbp(Xbal/PstI) 6uL

T7 vector(SpeI/PstI) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

2. Transformation

3. Ligation SD-Com again and transformation.

8.3

1. Picking colonies from T7-rbs-mbp_SD and mbp-Com, shaking at 37℃ for 10 hours.

2. Colonies PCR:

For 0.5uL

Rev 0.5uL

Easymix 5uL

template 0.2uL

ddH2O up to 10 uL

Identification by Electrophoresis

Sequence.

(The result is confirmed to be incorrect.)

3. Do ligation of T7-rbs-merr and transformation again, using the new B0034.

8.4

1. Picking colonies of T7-rbs-merr and shaking at 37℃ overnight.

2. Miniprep of T7-rbs-merr

3.Double Digestion and identification by Electrophoresis

EcoRI Buffer 1uL

EcorRI 0.75uL

PstI 0.75uL

T7-rbs-merr 7.5uL

Sequence.

8.5-8.8

Attend the iGEM-China meet up in Shang Hai!

8.11

The sequence of T7-rbs-merr is correct.

The sequence of mbp is correct.

The sequence of mbp-Com is correct

1. Positive transformation of mbp

2. DNA double digestion of mbp fragments for 1 hour:

Buffer3 2uL

Xbal 1.5uL

PstI 1.5uL

Product 10uL

ddH2O up to 20 uL

2. ligation of mbp fragments into plasmid vector for 1 hour

mbp (Xbal/PstI) 6uL

B0034 (s/p) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

3. Transformation

8.13

Test the mercury inductive expression system.

8.15

1. Picking 15 colonies from rbs-mbp_SD, shaking at 37℃ for 10 hours.

2. Colonies PCR:

For 0.5uL

Rev 0.5uL

Easymix 5uL

template 0.2uL

ddH2O up to 10 uL

Identification by Electrophoresis.

Sequence.

3. DNA double digestion of rbs-mbp fragments for 1 hour:

Buffer3 2uL

Xbal 1.5uL

PstI 1.5uL

Product 10uL

ddH2O up to 20 uL

4. ligation of mbp fragments into plasmid T7 vector for 1 hour

rbs-mbp (Xbal/PstI) 6uL

T7 vector (s/p) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

5. Transformation

Start the work of Human Practice

8.17

1. Picking 6 colonies from T7-rbs-mbp_SD, shaking at 37℃ for 10 hours.

2. Colonies PCR:

For 0.5uL

Rev 0.5uL

Easymix 5uL

template 0.2uL

ddH2O up to 10 uL

Identification by Electrophoresis.

Sequence.

8.18-8.20

Edit the Human practice questionnaire.

8.21

The sequence of T7-rbs-mbp_SD is correct

1. Positive transformation of T7-rbs-mbp_SD into BL-21a

2. DNA double digestion of T7-rbs-mbp fragments for 1 hour:

EcorRI Buffer 2uL

EcoRI 1.5uL

SpeI 1.5uL

Product 10uL

ddH2O up to 20 uL

3. Ligation of fragments into plasmid 1-23L(terminator) for 1 hour

T7-rbs-mbp (EcoRI/ SpeI) 6uL

1-23L (EcoRI/Xbal) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

4. Transformation

8.22

1. Picking colonies from T7-rbs-mbp-Terminator, shaking at 37℃ for 10 hours.

2. Colonies PCR:

For 0.5uL

Rev 0.5uL

Easymix 5uL

template 0.2uL

ddH2O up to 10 uL

Identification by Electrophoresis.

8.23

T7-rbs-mbp-Terminator: Digestion and identification by Electrophoresis

EcoRI Buffer 1uL

EcorRI 0.75uL

PstI 0.75uL

Product 7.5uL

Sequence.

8.24

Get the plasmid of Lpp-OmpA-mbp and start the assembly work.

1. DNA double digestion of T7-rbs-mbp-Terminator fragments for 1 hour:

EcorRI Buffer 2uL

EcoRI 1.5uL

SpeI 1.5uL

Product 10uL

ddH2O up to 20 uL

2. DNA double digestion of Lpp-OmpA-mbp for 1 hour:

EcorRI Buffer 2uL

EcoRI 1.5uL

XbaI 1.5uL

Product 10uL

ddH2O up to 20 uL

3. Ligation for 1 hour:

T7-rbs-mbp -terminator (EcoRI/ SpeI) 6uL

LOM (EcoRI/Xbal) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1uL

4. Transformation

8.25

Start the function test of mbp: inductive expression by IPTG

8.26

The sequence of T7-rbs-merr-terminator is correct.

Assembly plasmids: Digestion and identification by Electrophoresis

EcoRI Buffer 1uL

EcorRI 0.75uL

PstI 0.75uL

Product 7.5uL

Sequence.

September & October

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9.1-10.1

Pass the bench work to other guys.

1. Finished the wiki of Cytosol Expression.

2. Focus on the human practice work:

Lab contact and PI interviews.

Questionnaires sent out to NIBS, PKU, PUHSC.

Dig information out of books and reports

10.4

Chang Backbone of mbp and T7-rbs-mbp-Terminator:

1. Double digestion: inserts (E/S)

2. Electrophoresis in 1% agarose gel.

3. Excise the gel slice and extract target fragments.

4.Ligate inserts to pSB1C3 (E/S)

5. Transformation

10.5

1. Miniprep

2. Identification by Electrophoresis.

10.6-10.13

1. Writing the essay of Human practice.

2. Writing the Biosafety question required by iGEM committee.

10.14

Chang Backbone of rbs-mbp:

1. Double digestion: inserts (E/P)

2. Electrophoresis in 1% agarose gel.

3. Excise the gel slice and extract target fragments.

4.Ligate inserts to pSB1C3 (E/P)

5. Transformation

10.15

1. Picking colonies from rbs, shaking at 37℃ for 10 hours.

2. Colonies PCR:

For 0.5uL

Rev 0.5uL

Easymix 5uL

template 0.2uL

ddH2O up to 10 uL

Identification by Electrophoresis.

3. Digestion:

Buffer3 1uL

BamHI 0.75uL

rbs-mbp 5uL

ddH2O up to 10uL

identification by Electrophoresis

Sequence.

10.18

The sequence resuLt of mbp (pSB1C3) is correct.

The bench work is finished!

10.19-10.26

Revise the wiki of human practice.

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