Team:Peking/Notebook/MChen

From 2010.igem.org

(Difference between revisions)
(July)
Line 17: Line 17:
<div id="notebook home">
<div id="notebook home">
-
<div id="middlegreen">
+
<div id="middlegreen"><font color=#ffffff>
</html>
</html>
I characterized the CrtEBI (lycopene gene) biobrick K274100 submitted by team Cambridge 2009, for which two new biobricks was constructed. Addtionally, I contributed to the bioreporters partly, such as bioreporters for mercury or lead.  
I characterized the CrtEBI (lycopene gene) biobrick K274100 submitted by team Cambridge 2009, for which two new biobricks was constructed. Addtionally, I contributed to the bioreporters partly, such as bioreporters for mercury or lead.  
Line 98: Line 98:
2.
2.
Add 10μl ddH2O, leave the water in the well for 30 sec(red).
Add 10μl ddH2O, leave the water in the well for 30 sec(red).
 +
 +
BBa_K118014 rbs+crtE 2-18H/2010 pSB1A2
-
        BBa_K118014 rbs+crtE 2-18H/2010 pSB1A2
+
BBa_K118013 rbs+crtY 2-18F/2010 pSB1A2
-
        BBa_K118013 rbs+crtY 2-18F/2010 pSB1A2
+
BBa_K118006 rbs+crtB 2-16N/2010 pSB1A2
-
        BBa_K118006 rbs+crtB 2-16N/2010 pSB1A2
+
BBa_K118005 rbs+crtI 2-16L/2010 pSB1A2
-
        BBa_K118005 rbs+crtI 2-16L/2010 pSB1A2
+
BBa_K274003 VioABDE 3-20H/2010 pSB1K3
-
        BBa_K274003 VioABDE 3-20H/2010 pSB1K3
+
BBa_K274004 VioABCE 3-20J/2010 pSB1K3
-
 
+
-
        BBa_K274004 VioABCE 3-20J/2010 pSB1K3
+
2. Pipette 2μl of the resuspended DNA transform into the Trans 5a competent cells.
2. Pipette 2μl of the resuspended DNA transform into the Trans 5a competent cells.
-
        Each tube: Competent cells 50μl + DNA 2μl
+
Each tube: Competent cells 50μl + DNA 2μl
3. Picking a single colony and inoculate for 15 hours(in 5ml antibiotics culture)
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)
4. Miniprep and use spectrophotometer to estimate the concentration of DNA(50×dilution)
 +
 +
VioABDE A260=0.027 conc=1.3401μg/ml
-
        VioABDE A260=0.027 conc=1.3401μg/ml
+
VioABCE A260=0.022 conc=1.0782μg/ml
-
        VioABCE A260=0.022 conc=1.0782μg/ml
+
CrtY    A260=0.047 conc=2.3349μg/ml
-
        CrtY   A260=0.047 conc=2.3349μg/ml
+
CrtI   A260=0.078 conc=3.8963μg/ml
-
        CrtI   A260=0.078 conc=3.8963μg/ml
+
CrtZ   A260=0.044 conc=2.2146μg/ml
-
        CrtZ   A260=0.044 conc=2.2146μg/ml
+
CrtE   A260=0.087 conc=4.3482μg/ml
-
        CrtE    A260=0.087 conc=4.3482μg/ml
+
CrtB    A260=0.055 conc=2.7299μg/ml
-
 
+
-
        CrtB    A260=0.055 conc=2.7299μg/ml
+
5. PCR for VioA, VioB, VioC, VioD, VioE
5. PCR for VioA, VioB, VioC, VioD, VioE
-
      The primer for VioA, VioB, VioC, VioD and VioE are:  
+
The primer for VioA, VioB, VioC, VioD and VioE are:  
 +
 
ViolaA_For ccggaattcgcggccgcttctagATGAAACATTCTTCCGATATCTGCATTGTTGG
ViolaA_For ccggaattcgcggccgcttctagATGAAACATTCTTCCGATATCTGCATTGTTGG
 +
ViolaA_Rev aaactgcagcggccgctactagtaTCACGCGGCGATACGCTGCA
ViolaA_Rev aaactgcagcggccgctactagtaTCACGCGGCGATACGCTGCA
 +
ViolaB_For ccggaattcgcggccgcttctagATGAGCATTCTGGATTTCCCGCGTATC
ViolaB_For ccggaattcgcggccgcttctagATGAGCATTCTGGATTTCCCGCGTATC
 +
ViolaB_Rev aaactgcagcggccgctactagtaTTAGGCCTCGCGGCTCAGTTTG
ViolaB_Rev aaactgcagcggccgctactagtaTTAGGCCTCGCGGCTCAGTTTG
 +
ViolaC_For ccggaattcgcggccgcttctagATGAAACGTGCGATTATCGTTGG
ViolaC_For ccggaattcgcggccgcttctagATGAAACGTGCGATTATCGTTGG
 +
ViolaC_Rev aaactgcagcggccgctactagtaTCAATTCACGCGACCAATCTTG
ViolaC_Rev aaactgcagcggccgctactagtaTCAATTCACGCGACCAATCTTG
 +
ViolaD_For ccggaattcgcggccgcttctagATGAAGATTCTGGTCATTGGTG
ViolaD_For ccggaattcgcggccgcttctagATGAAGATTCTGGTCATTGGTG
 +
ViolaD_Rev aaactgcagcggccgctactagtaTCAGCGCTGCAAAGCATAAC
ViolaD_Rev aaactgcagcggccgctactagtaTCAGCGCTGCAAAGCATAAC
 +
  ViolaE_For      ccg gaattc gcggccgct tctagATGGAGAACCGTGAGCCAC  
  ViolaE_For      ccg gaattc gcggccgct tctagATGGAGAACCGTGAGCCAC  
 +
ViolaE_Rev      aaa ctgcag cggccgct actagtaTTAGCGCTTGGCCGCGAAA   
ViolaE_Rev      aaa ctgcag cggccgct actagtaTTAGCGCTTGGCCGCGAAA   
-
dd                     11.7μl
+
dd H2O                    11.7μl
 +
 +
5×phusion HF buffer            4μl
-
      5×phusion HF buffer            4μl
+
2.5MdNTP                      1.6μl
-
      2.5MdNTP                      1.6μl
+
For                            1μl
-
      For                             1μl
+
Rev                             1μl
-
      Rev                            1μl
+
Template                      0.5μl
-
      Template                      0.5μl
+
(chill on ice)
-
                                          (chill on ice)
+
Phusion pol                    0.2μl
-
 
+
-
      Phusion pol                    0.2μl
+
Line 170: Line 180:
-
7.8-7.14
+
===7.8-7.14===
1. Double digest for CrtE、CrtB、CrtI、CrtY、CrtZ
1. Double digest for CrtE、CrtB、CrtI、CrtY、CrtZ
-
      CrtB、CrtY、CrtZ: EcoRI and XbaI
+
CrtB、CrtY、CrtZ: EcoRI and XbaI
CrtE、CrtI: EcoRI and SpeI
CrtE、CrtI: EcoRI and SpeI
Line 182: Line 192:
NEB:  
NEB:  
-
      CrtE or CrtI            10μl
+
CrtE or CrtI            10μl
-
      10×EcoRI buffer      2μl
+
10×EcoRI buffer      2μl
-
      EcoRI                  1μl
+
EcoRI                  1μl
-
      SpeI                    1μl
+
SpeI                    1μl
-
      ddH2O                6μl
+
ddH2O                6μl
-
      Total                  20μl
+
Total                  20μl
-
    Takara:
+
Takara:
-
      CrtB or CrtY or CrtZ      10μl
+
CrtB or CrtY or CrtZ      10μl
-
      10×buffer(1×M)        2μl
+
EcoRI                  1μl
-
      EcoRI                  1μl
+
XbaI                    1μl
-
      XbaI                    1μl
+
ddH2O                6μl
-
      ddH2O                6μl
+
Total                  20μl
-
      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.
-
 
+
-
      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.
+
Line 218: Line 225:
2. Ligate CrtE and CrtB
2. Ligate CrtE and CrtB
-
          CrtB            2μl
+
CrtB            2μl
-
          CrtE            6μl
+
CrtE            6μl
-
          10×T4 buffer  1μl
+
10×T4 buffer  1μl
-
          T4 DNA Ligase    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.
3. Transform CrtEB, pick five single colonies and inoculate(in 5ml antibiotics culture), miniprep by Trans kit, identified by XbaI and PstI digestion.
 +
4.
4.
-
NEB:  
+
NEB:  
-
      CrtEB                  5μl
+
CrtEB                  5μl
-
      10×3 buffer            2μl
+
10×3 buffer            2μl
-
      XbaI                    1μl
+
XbaI                    1μl
-
      PstI                    1μl
+
PstI                    1μl
-
      ddH2O                  11μl
+
ddH2O                  11μl
-
      Total                    20μl
+
Total                    20μl
5. Sequence by Hua Da company. Two results were right
5. Sequence by Hua Da company. Two results were right
-
6.
+
-
7. 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.  
+
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.
-
8.
+
-
9. Gradient PCR for VioB and VioE
+
-
10.
+
-
        Easymix              10μl
+
-
        ddH2O              8.5μl
+
7. Gradient PCR for VioB and VioE
 +
 +
Easymix              10μl
-
        Primer For            0.5μl
+
ddH2O              8.5μl
-
        Primer Rev           0.5μl
+
Primer For           0.5μl
-
        Template              0.5μl
+
Primer Rev            0.5μl
-
        Total                20μl
+
Template              0.5μl
-
    Gradient:  
+
Total                20μl
 +
 
 +
Gradient:  
5  50.4℃
5  50.4℃
Line 278: Line 286:
6.  PCR for VioB by taq polymerase
6.  PCR for VioB by taq polymerase
-
      TransEasymix              10μl
+
TransEasymix              10μl
-
      ddH2O              8.5μl
+
ddH2O              8.5μl
-
      Primer For            0.5μl
+
Primer For            0.5μl
-
      Primer Rev            0.5μl
+
Primer Rev            0.5μl
-
      Template              0.5μl(VioABDE)
+
Template              0.5μl(VioABDE)
-
      Total                20μl
+
Total                20μl
Gradient:  
Gradient:  
Line 310: Line 318:
1. Transform VioABCDE, double digestion VioABCDE for VioC(BamHI, BglII)
1. Transform VioABCDE, double digestion VioABCDE for VioC(BamHI, BglII)
-
    Takara:
+
Takara:
-
      VioABCDE                          10μl
+
VioABCDE                          10μl
-
      BamHI                            1.5μl  
+
BamHI                            1.5μl  
-
      BglII                              1.5μl
+
BglII                              1.5μl
-
      10×K buffer                      2μl
+
10×K buffer                      2μl
-
      ddH2O                            5μl
+
ddH2O                            5μl
-
      total                              20μl
+
total                              20μl
-
  Control
+
Control
-
      VioABCDE                          10μl
+
VioABCDE                          10μl
-
      BamHI                              2μl  
+
BamHI                              2μl  
-
      10×K buffer                        2μl
+
10×K buffer                        2μl
-
      ddH2O                            6μl
+
ddH2O                            6μl
-
      total                              20μl
+
total                              20μl
Line 344: Line 352:
37℃ overnight
37℃ overnight
-
  Electrophoresis in 1.5% agarose gel, the result of BamHI digestion was the same as double digestion.
+
Electrophoresis in 1.5% agarose gel, the result of BamHI digestion was the same as double digestion.
2. Gradient PCR for VioC
2. Gradient PCR for VioC
Line 350: Line 358:
Easymix              10μl
Easymix              10μl
-
      ddH2O              8.5μl
+
ddH2O              8.5μl
 +
 +
Primer For            0.5μl
-
      Primer For           0.5μl
+
Primer Rev           0.5μl
-
      Primer Rev            0.5μl
+
Template              0.5μl(VioABCDE)
-
      Template              0.5μl(VioABCDE)
+
Total                20μl
-
 
+
-
      Total                20μl
+
Gradient
Gradient
Line 394: Line 402:
3. different reaction systems
3. different reaction systems
-
  (1)
+
(1)
-
          XP CrtEBIY(insert)            2μl    1μl  4μl
+
XP CrtEBIY(insert)            2μl    1μl  4μl
-
          SP terminator(vector)            6μl    7μl  4μl
+
SP terminator(vector)            6μl    7μl  4μl
-
          10×T4 buffer  1μl
+
10×T4 buffer  1μl
-
          T4 DNA Ligase  1μl
+
T4 DNA Ligase  1μl
-
                Total    10μl
+
Total    10μl
-
  (2)
+
(2)
-
          XP CrtEBIY(insert)            4μl    2μl  8μl
+
XP CrtEBIY(insert)            4μl    2μl  8μl
-
          SP terminator(vector)          12μl  14μl  8μl
+
SP terminator(vector)          12μl  14μl  8μl
-
          10×T4 buffer  2μl
+
10×T4 buffer  2μl
-
          T4 DNA Ligase  2μl
+
T4 DNA Ligase  2μl
-
                Total    20μl
+
Total    20μl
-
4. different temperatures(16℃ and 25℃).
+
4. different temperatures(16℃ and 25℃).
Line 424: Line 432:
The results were on our wiki, so I didn’t repeat here.
The results were on our wiki, so I didn’t repeat here.
-
 
+
==August==
<html>
<html>

Revision as of 08:11, 26 October 2010




   Mei Chen's Notes
                                                                                                                                                goto her page
I characterized the CrtEBI (lycopene gene) biobrick K274100 submitted by team Cambridge 2009, for which two new biobricks was constructed. Addtionally, I contributed to the bioreporters partly, such as bioreporters for mercury or lead.


download her notes

Contents


July

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

[TOP]

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