Team:Peking/Notebook/MJing

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   Jing Miao's Notes
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I designed to engineer this operon under the regulation of T7 promoter, which is regulated by upstream T7 polymerase, to fulfill the goal that the absorbance of Hg(II) will be enhanced for an efficient bioabsorbent. Also I cloned and assembled agn43 into the inductive aggregation module. Agn43 is drove by PO promoter, which is the terminal of a cascade amplification


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Contents

Contents


July

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[TOP]

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

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