Team:Peking/Notebook/MChen

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Contents

• July, 2010

• August, 2010

• September & October, 2010

July

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7.1-7.7

I wanted to get CrtE, CrtB, CrtI, CrtY, CrtZ, VioA, VioB, VioC, VioD and VioE for producing CrtEBI, CrtEBIY, CrtEBIYZ, VioABCE, VioABDE and VioABCDE.

1. Extract DNA from the registry 2. Add 10μl ddH2O, leave the water in the well for 30 sec(red).

BBa_K118014 rbs+crtE 2-18H/2010 pSB1A2

BBa_K118013 rbs+crtY 2-18F/2010 pSB1A2

BBa_K118006 rbs+crtB 2-16N/2010 pSB1A2

BBa_K118005 rbs+crtI 2-16L/2010 pSB1A2

BBa_K274003 VioABDE 3-20H/2010 pSB1K3

BBa_K274004 VioABCE 3-20J/2010 pSB1K3

2. Pipette 2μl of the resuspended DNA transform into the Trans 5a competent cells.

Each tube: Competent cells 50μl + DNA 2μl

3. Picking a single colony and inoculate for 15 hours(in 5ml antibiotics culture)

4. Miniprep and use spectrophotometer to estimate the concentration of DNA(50×dilution)

VioABDE A260=0.027 conc=1.3401μg/ml

VioABCE A260=0.022 conc=1.0782μg/ml

CrtY A260=0.047 conc=2.3349μg/ml

CrtI A260=0.078 conc=3.8963μg/ml

CrtZ A260=0.044 conc=2.2146μg/ml

CrtE A260=0.087 conc=4.3482μg/ml

CrtB A260=0.055 conc=2.7299μg/ml

5. PCR for VioA, VioB, VioC, VioD, VioE

The primer for VioA, VioB, VioC, VioD and VioE are:

ViolaA_For ccggaattcgcggccgcttctagATGAAACATTCTTCCGATATCTGCATTGTTGG

ViolaA_Rev aaactgcagcggccgctactagtaTCACGCGGCGATACGCTGCA

ViolaB_For ccggaattcgcggccgcttctagATGAGCATTCTGGATTTCCCGCGTATC

ViolaB_Rev aaactgcagcggccgctactagtaTTAGGCCTCGCGGCTCAGTTTG

ViolaC_For ccggaattcgcggccgcttctagATGAAACGTGCGATTATCGTTGG

ViolaC_Rev aaactgcagcggccgctactagtaTCAATTCACGCGACCAATCTTG

ViolaD_For ccggaattcgcggccgcttctagATGAAGATTCTGGTCATTGGTG

ViolaD_Rev aaactgcagcggccgctactagtaTCAGCGCTGCAAAGCATAAC

ViolaE_For ccg gaattc gcggccgct tctagATGGAGAACCGTGAGCCAC

ViolaE_Rev aaa ctgcag cggccgct actagtaTTAGCGCTTGGCCGCGAAA

dd H2O 11.7μl

5×phusion HF buffer 4μl

2.5MdNTP 1.6μl

For 1μl

Rev 1μl

Template 0.5μl

(chill on ice)

Phusion pol 0.2μl

I did the PCR three times, VioB, VioC and VioE hadn’t result. VioA and VioD were extracted from the gel.

7.8-7.14

1. Double digest for CrtE、CrtB、CrtI、CrtY、CrtZ

CrtB、CrtY、CrtZ: EcoRI and XbaI

CrtE、CrtI: EcoRI and SpeI

NEB:

CrtE or CrtI 10μl

10×EcoRI buffer 2μl

EcoRI 1μl

SpeI 1μl

ddH2O 6μl

Total 20μl

Takara:

CrtB or CrtY or CrtZ 10μl

EcoRI 1μl

XbaI 1μl

ddH2O 6μl

Total 20μl

After double digestion, electrophoresis in 1.5% agarose gel to test the results and the result of CrtI was wrong. I did the double digestion for CrtI again and the result was wrong again.

2. Ligate CrtE and CrtB

CrtB 2μl

CrtE 6μl

10×T4 buffer 1μl

T4 DNA Ligase 1μl

3. Transform CrtEB, pick five single colonies and inoculate(in 5ml antibiotics culture), miniprep by Trans kit, identified by XbaI and PstI digestion.

4. NEB:

CrtEB 5μl

10×3 buffer 2μl

XbaI 1μl

PstI 1μl

ddH2O 11μl

Total 20μl

5. Sequence by Hua Da company. Two results were right

6. Transform BBa_K118005 rbs+crtI and double digestion again, but the result was still wrong. The idea for producing CrtEBI, CrtEBIY and CrtEBIYZ by myself was given up because of the time limition.

7. Gradient PCR for VioB and VioE

Easymix 10μl

ddH2O 8.5μl

Primer For 0.5μl

Primer Rev 0.5μl

Template 0.5μl

Total 20μl

Gradient:

5 50.4℃

6 53.0℃

7 55.8℃

8 58.5℃

9 61.0℃

Electrophoresis in 1.5% agarose gel to test the results of PCR, VioE had results.

6. PCR for VioB by taq polymerase

TransEasymix 10μl

ddH2O 8.5μl

Primer For 0.5μl

Primer Rev 0.5μl

Template 0.5μl(VioABDE)

Total 20μl

Gradient:

5 52.5℃

6 55.1℃

7 57.8℃

8 60.5℃

9 63.0℃

Electrophoresis in 1.5% agarose gel to test the results of PCR VioB, five gradients had results. All were extracted from the gel and sequence, gradient 6 and gradient 7 had no mutation.

7.15-7.22

1. Transform VioABCDE, double digestion VioABCDE for VioC(BamHI, BglII)

Takara:

VioABCDE 10μl

BamHI 1.5μl

BglII 1.5μl

10×K buffer 2μl

ddH2O 5μl

total 20μl

Control

VioABCDE 10μl

BamHI 2μl

10×K buffer 2μl

ddH2O 6μl

total 20μl

37℃ 4 hours

Or

37℃ overnight

Electrophoresis in 1.5% agarose gel, the result of BamHI digestion was the same as double digestion.

2. Gradient PCR for VioC

Easymix 10μl

ddH2O 8.5μl

Primer For 0.5μl

Primer Rev 0.5μl

Template 0.5μl(VioABCDE)

Total 20μl

Gradient

5 46.4℃

6 49℃

7 51.8℃

8 54.5℃

9 57℃

Electrophoresis in 1.5% agarose gel, no result.

I did the transformation, double digestion and PCR twice but the result was the same. The idea for producing VioABCE, VioABDE and VioABCDE by myself was given up because of the time limition.

7.23-8.17

I began to do the characterization for CrtEBI(K274100) and CrtEBIY(K274200).

CrtEBI(K274100) and CrtEBIY(K274200) were ligated to constitutive promoter (BBa_J23103) and terminator (B0015). All the protocols followed protocols above. Unfortunately, I failed in ligating the CrtEBIY and terminator (B0015) though I did this experiment many times in different conditions. The conditions included

1. ligase with different companies(NEB and Trans)

2. different ligation time(1, 2, 4, 12 hours)

3. different reaction systems

(1)

XP CrtEBIY(insert) 2μl 1μl 4μl

SP terminator(vector) 6μl 7μl 4μl

10×T4 buffer 1μl

T4 DNA Ligase 1μl

Total 10μl

(2)

XP CrtEBIY(insert) 4μl 2μl 8μl

SP terminator(vector) 12μl 14μl 8μl

10×T4 buffer 2μl

T4 DNA Ligase 2μl

Total 20μl

4. different temperatures(16℃ and 25℃).

The results were on our wiki, so I didn’t repeat here.

August

[TOP]

8.18-8.25

After the characterization of CrtEBI(K274100) and CrtEBIY(K274200), we chose CrtEBI as our reporter. So I began to ligate the reporter to the promoter.

1. Double digest for CrtEBI and PmerT(on pSB1C3, submitted by Yiwei Chen)

NEB:

CrtEBI 10μl

10×3 buffer 2μl

XbaI 1μl

PstI 1μl

ddH2O 6μl

Total 20μl

PmerT 10μl

10×2 buffer 2μl

SpeI 1μl

PstI 1μl

ddH2O 6μl

Total 20μl

37℃ overnight

2. Ligate

XP CrtEBI(insert) 2μl 1μl 4μl

SP PmerT (vector) 6μl 7μl 4μl

10×T4 buffer 1μl

T4 DNA Ligase 1μl

Total 10μl

16℃ overnight(NEB)

Or

25℃ 2 hours(Trans)

3. Transform and pick five single colonies and inoculate (in 5ml antibiotics culture), miniprep, digest and sequence.

This process was repeated many times in different ligation conditions, but all failed.

8.26-9.10

I used another strategy. Two strands of the promoter PmerT and PpbrA which were synthesized by Hua Da company was used. The two strands included EcoRI and SpeI site. The promoters were inserted to the vector which has CrtEBI.

1. Anneal

Mer_pro_for 1.5μl

Mer_pro_rev 1.5μl

PpbrA_for 1.5μl

PpbrA_rev 1.5μl

Metalbath 95℃ 5min

Cool to room temperature(on metalbath)

2. Phosphorylation

Insert 3μl

T4-Ligase buffer(NEB) 1μl

T4 polykinase(NEB) 1μl

ddH2O 4μl

total 9μl

37℃ 30min

3. Ligation

Insert(after phosphorylation) 9μl

T4-ligase 1μl

Vector 1μl

Total 11μl

16℃ 1hour

Or

16℃ overnight

4. Transform, pick colonies, miniprep, digest and sequence.

After repeated several times, I succeed in PpbrA-CrtEBI at last. Then I ligated the CrtEBI to PmerT in pSB1C3 again and succeed. But the worst was still to come. The E.coli which has PpbrA-CrtEBI or PmerT- CrtEBI appeared bright red, just as the constitutive promoter-CrtEBI. So we had to give up CrtEBI as our repoter.

September & October

9.1-9.10

9.10-10.10

The summer vocation has come to a close. I retarded the progress. All the protocols followed protocols above.

1. Three colones

Pmert-pag

PmerT-ogr

PmerT-phiR73delta

Everything went off without a hitch.

2. Change backbones

constitutive promoter(BBa_J23103)-CrtEBI

terminator (B0015) )-CrtEBI

constitutive promoter(BBa_J23103)-CrtEBIY

The double digestion (EcoRI and PstI) for constitutive promoter(BBa_J23103)-CrtEBIY failed twice. The double digestion (EcoRI and PstI) result was the same as the EcoRI digestion or PstI digestion. The terminator (B0015) )-CrtEBI transformed twice after changing the backbone, but the results of sequence were all wrong.