Team:Peking/Notebook/MChen

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   Mei Chen's Notes
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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.


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Contents

July

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 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
      10×buffer(1×M)        2μl
      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. 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. 8. 9. Gradient PCR for VioB and VioE 10.

       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.