Team:Peking/Notebook/MChen

From 2010.igem.org

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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 32: Line 32:
* <span style="font-size:4mm;">[[Team:Peking/Notebook/MChen#August| August, 2010]]</span>
* <span style="font-size:4mm;">[[Team:Peking/Notebook/MChen#August| August, 2010]]</span>
-
* <span style="font-size:4mm;">[[Team:Peking/Notebook/MChen#September| September, 2010]]</span>
+
* <span style="font-size:4mm;">[[Team:Peking/Notebook/MChen#September & October| September & October, 2010]]</span>
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+
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/MChen#October| October, 2010]]</span>
+
-
 
+
-
 
+
==July==
==July==
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|-  
|-  
|}
|}
-
[[https://2010.igem.org/Team:Peking/Notebook/MChen TOP]]
+
[<html><a href="#top">TOP</a></html>]
===7.1-7.7===
===7.1-7.7===
Line 98: Line 94:
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_Rev     aaa ctgcag cggccgct actagtaTTAGCGCTTGGCCGCGAAA 
+
ViolaE_For     ccg gaattc gcggccgct tctagATGGAGAACCGTGAGCCAC
-
dd                      11.7μl
 
-
      5×phusion HF buffer            4μl
+
ViolaE_Rev      aaa ctgcag cggccgct actagtaTTAGCGCTTGGCCGCGAAA 
 +
 
 +
dd H2O                    11.7μl
 +
 +
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 168: Line 174:
I did the PCR three times, VioB, VioC and VioE hadn’t result. VioA and VioD were extracted from the gel.
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===
-
 
+
-
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 186:
NEB:  
NEB:  
-
      CrtE or CrtI            10μl
+
CrtE or CrtI            10μl
-
 
+
-
      10×EcoRI buffer      2μl
+
-
 
+
-
      EcoRI                  1μl
+
-
      SpeI                    1μl
+
10×EcoRI buffer      2μl
-
      ddH2O                6μl
+
EcoRI                  1μl
-
      Total                  20μl
+
SpeI                    1μl
 +
ddH2O                6μl
 +
Total                  20μl
-
    Takara:
 
-
      CrtB or CrtY or CrtZ      10μl
 
-
      10×buffer(1×M)        2μl
+
Takara:
-
      EcoRI                  1μl
+
CrtB or CrtY or CrtZ      10μl
-
      XbaI                    1μl
+
EcoRI                  1μl
-
      ddH2O                6μl
+
XbaI                    1μl
-
      Total                  20μl
+
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.
+
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 219:
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 280:
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 302: Line 304:
9  63.0℃
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.
+
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===
===7.15-7.22===
Line 310: Line 310:
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 344:
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 350:
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 394:
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℃).
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==
 +
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"
 +
|-
 +
|style="text-align:center"| Mon
 +
|style="text-align:center"| Tue
 +
|style="text-align:center"| Wed
 +
|style="text-align:center"| Thu
 +
|style="text-align:center"| Fri
 +
|style="text-align:center"| Sat
 +
|style="text-align:center"| Sun
 +
|-
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|1]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|2]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|3]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|4]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|5]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|6]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|7]]
 +
|-
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|8]]
 +
|style="text-align:center"|[[Team:Peking/Notebook/MChen#7.23-8.17|9]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|10]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|11]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|12]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|13]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|14]]
 +
|-
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|15]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|16]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#7.23-8.17|17]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.18-8.25|18]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.18-8.25|19]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.18-8.25|20]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.18-8.25|21]]
 +
|-
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.18-8.25|22]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.18-8.25|23]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.18-8.25|24]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.18-8.25|25]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.26-9.10|26]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.26-9.10|27]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.26-9.10|28]]
 +
|-
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.26-9.10|29]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.26-9.10|30]]
 +
|style="text-align:center"| [[Team:Peking/Notebook/MChen#8.26-9.10|31]]
 +
|style="text-align:center"| -
 +
|style="text-align:center"| -
 +
|style="text-align:center"| -
 +
|style="text-align:center"| -
 +
|}
 +
[<html><a href="#top">TOP</a></html>]
 +
 +
===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==
 +
[[Team:Peking/Notebook/MChen#8.26-9.10|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.
 +

Latest revision as of 10:08, 27 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

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]

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.