Team:Peking/Notebook/YSheng

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[[https://2010.igem.org/Team:Peking/Notebook/YSheng TOP]]
[[https://2010.igem.org/Team:Peking/Notebook/YSheng TOP]]
 +
 +
===7.2 – 7.4===
 +
RBS(B0034)- merR construction
 +
 +
Vector Digestion:
 +
 +
B0034  plamid  5μL
 +
 +
SpeI(NEB)  1.5μL
 +
 +
PstI(NEB)    1.5μL
 +
 +
Buffer      2μL
 +
 +
ddH2O      10μL
 +
 +
Gel extraction the digested product
 +
 +
Ligation:
 +
 +
RBS-merR(digested by EcoRI & PstI)  3 μL  (prepared by Ao Liu)
 +
BBa_ B0034  (s.p. digested)  1 μL
 +
ddH2O  4 μL
 +
buffer  1 μL
 +
ligase  1 μL
 +
Transformed all the 10μL to trans 5a competent cell.
 +
 +
Colony Picking-up & PCR certification
 +
Taq Mix  5 μL
 +
Univ-For  0.5 μL
 +
Univ-Rev  0.5 μL
 +
ddH2O    4 μL
 +
miniprep 3 right clones and sequence them.
 +
 +
PmerT – GFP (E0840) construction
 +
The PmerT insert preparation: Two reverse and complementary strains of PmerT were synthesized with artificial sticky ends if they were annealed.
 +
 +
Vector Digestion:
 +
E0840 plamid  5 μL
 +
EcoR I(NEB)  1.5 μL
 +
XbaI(NEB)    1.5 μL
 +
EcoRI Buffer  2 μL
 +
ddH2O        10 μL
 +
Gel extraction the digested product
 +
 +
Annealing: 95℃ 5min, cooled done in metal bath.
 +
PmerT-For  5μL
 +
PmerT-Rev  5μL
 +
Phosphalation: 37℃ for 30min
 +
T4 PNK    1μL
 +
T4 ligase buffer  1μL
 +
ddH2O    4μL
 +
Annealing product  3μL
 +
 +
Ligation
 +
PmerT Phosphalation product  9μL
 +
Ligase  1μL
 +
E0840 vector (E. X. digested)  1μL
 +
Transformed all the 11μL to trans 5a competent cell.
 +
 +
Colony Picking-up & PCR certification
 +
Taq Mix    5 μL
 +
PmerT-For  0.5 μL
 +
E0840-Rev  0.5 μL
 +
ddH2O    4 μL
 +
miniprep 3 right clones and sequence them.
 +
===7.5-7.11===
 +
Pc(J23100) – RBS- merR construction
 +
Vector Digestion:
 +
J23100 vector digested by S.P.
 +
RBS-merR digested by X.P.
 +
Gel extraction of vector and insert
 +
 +
Ligation
 +
Transformed all the 10μL to trans 5a competent cell.
 +
 +
Colony Picking-up & PCR certification
 +
Primer: Univ-For & Univ-Rev
 +
miniprep 3 right clones and sequence them.
 +
 +
Changed the backbone of PmerT-GFP from pSB1A2 to pSB3K3
 +
Vector Digestion:
 +
pSB3K3 vector digested by S.P.
 +
RBS-merR insert digested by X.P.
 +
Gel extraction of vector and insert
 +
 +
Ligation
 +
6 μL insert + 2 μL vector
 +
Transformed all the 10μL to trans 5a competent cell.
 +
 +
Colony Picking-up & PCR certification
 +
Primer: PmerT-For & Univ-Rev
 +
miniprep 3 right clones
 +
===7.12-7.18===
 +
Reconstruct Pc(J23100) – RBS- merR construction since the sequence results were wrong.
 +
 +
Made competent bacteria containing the PmerT-GFP(pSB3K3)
 +
Cultured the bacteria in LB overnight
 +
Dilute the bacteria 1:100 with LB and culture it until OD reached 0.4-0.6
 +
Centrifuge 500μL bacteria 5000rpm for 5min
 +
Resuspended the cell with 500μL chilled 0.1M CaCl2, incubated in ice for 30min
 +
Centrifuge 500μL bacteria 5000rpm for 5min
 +
Resuspended the cell with 100μL chilled 0.1M CaCl2
 +
 +
Constructed strains with both PmerT-GFP(pSB3K3) and Pc(1-18I)-RBS-MerR(pSB1A2)
 +
Transformation the competent bacteria of PmerT-GFP(pSB3K3) with 2μL Pc(1-18I)-RBS-MerR(pSB1A2) plasmid.
 +
 +
===7.19-7.25===
 +
Inducement of the strain with two plasmids which were constructed last week
 +
7.20
 +
With Hg(II)’s concentration = 1E-6 mol/L, 1E-7 mol/L and 1E-8 mol/L to certify that the strain worked.
 +
7.22
 +
With Hg(II)’s concentration = 1E-10, 1E-9, 1E-8, 2E-8, 4E-8, 6E-8, 8E-8, 1E-7, 1E-6, 1E-5(mol/L)
 +
3 duplicates
 +
GFP’s intensity was measured by microplate reader.
 +
7.23
 +
With Hg(II)’s concentration = 1E-10, 1E-9, 1E-8, 4E-8, 7E-8, 1E-7, 4E-7, 7E-7, 1E-6, 1E-5(mol/L)
 +
3 duplicates
 +
7.24
 +
With Hg(II)’s concentration = 1E-10, 1E-9, 1E-8, 4E-8, 7E-8, 1E-7, 4E-7, 7E-7, 1E-6, 1E-5(mol/L
 +
The OD was measured by Spectrophotometer to conform Hg(II)’s effect on bacterial growth
 +
 +
7.25
 +
With Hg(II)’s concentration = 1E-10, 1E-9, 4E-9, 1E-8, 1.5E-8, 2E-8, 3E-8, 4E-8, 7E-8, 1E-7, 1.5E-7, 2E-7, 3E-7, 4E-7, 7E-7, 1E-6, 4E-6, 1E-5(mol/L)
 +
OD600 of each sample was also measured by Spectrophotometer.
 +
 +
===7.26-8.2===
 +
Measurement of the 3 – dimensional figure which represented the time and dose response curve to mercury both for OD600 and GFP intensity.
 +
 +
I first tried to sample 100μL every 20min to the black-96-well plate and stored the plate in 4℃，OD was measured by spectrophotometer right after sampling.
 +
 +
The optimized protocol was to incubate the bacteria with Hg(II) in the microplate (black one for GFP intensity measurement and transparent one for OD600 measurement), then the two plates were measured by the microplate reader every 20min. During the interval of measurement, the two plates were cultured in the 37℃ shaker.

Revision as of 06:43, 26 October 2010





   Yin Shen's Notes
                                                                                                                                                goto her page
My work mainly focuses on the characterization of merOP. The response curve of merOP was characterized in Ecoli strains possessing different genetic component combinations to figure out the affects of different module to merOP and finally contributed to the rational design of the bioreporter.

download her notes

Contents

July

Mon Tue Wed Thu Fri Sat Sun
- - - - 1 3 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]

7.2 – 7.4

RBS(B0034)- merR construction

Vector Digestion:

B0034 plamid 5μL

SpeI(NEB) 1.5μL

PstI(NEB) 1.5μL

Buffer 2μL

ddH2O 10μL

Gel extraction the digested product

Ligation:

RBS-merR(digested by EcoRI & PstI) 3 μL (prepared by Ao Liu) BBa_ B0034 (s.p. digested) 1 μL ddH2O 4 μL buffer 1 μL ligase 1 μL Transformed all the 10μL to trans 5a competent cell.

Colony Picking-up & PCR certification Taq Mix 5 μL Univ-For 0.5 μL Univ-Rev 0.5 μL ddH2O 4 μL miniprep 3 right clones and sequence them.

PmerT – GFP (E0840) construction The PmerT insert preparation: Two reverse and complementary strains of PmerT were synthesized with artificial sticky ends if they were annealed.

Vector Digestion: E0840 plamid 5 μL EcoR I(NEB) 1.5 μL XbaI(NEB) 1.5 μL EcoRI Buffer 2 μL ddH2O 10 μL Gel extraction the digested product

Annealing: 95℃ 5min, cooled done in metal bath. PmerT-For 5μL PmerT-Rev 5μL Phosphalation: 37℃ for 30min T4 PNK 1μL T4 ligase buffer 1μL ddH2O 4μL Annealing product 3μL

Ligation PmerT Phosphalation product 9μL Ligase 1μL E0840 vector (E. X. digested) 1μL Transformed all the 11μL to trans 5a competent cell.

Colony Picking-up & PCR certification Taq Mix 5 μL PmerT-For 0.5 μL E0840-Rev 0.5 μL ddH2O 4 μL miniprep 3 right clones and sequence them.

7.5-7.11

Pc(J23100) – RBS- merR construction Vector Digestion: J23100 vector digested by S.P. RBS-merR digested by X.P. Gel extraction of vector and insert

Ligation Transformed all the 10μL to trans 5a competent cell.

Colony Picking-up & PCR certification Primer: Univ-For & Univ-Rev miniprep 3 right clones and sequence them.

Changed the backbone of PmerT-GFP from pSB1A2 to pSB3K3 Vector Digestion: pSB3K3 vector digested by S.P. RBS-merR insert digested by X.P. Gel extraction of vector and insert

Ligation 6 μL insert + 2 μL vector Transformed all the 10μL to trans 5a competent cell.

Colony Picking-up & PCR certification Primer: PmerT-For & Univ-Rev miniprep 3 right clones

7.12-7.18

Reconstruct Pc(J23100) – RBS- merR construction since the sequence results were wrong.

Made competent bacteria containing the PmerT-GFP(pSB3K3) Cultured the bacteria in LB overnight Dilute the bacteria 1:100 with LB and culture it until OD reached 0.4-0.6 Centrifuge 500μL bacteria 5000rpm for 5min Resuspended the cell with 500μL chilled 0.1M CaCl2, incubated in ice for 30min Centrifuge 500μL bacteria 5000rpm for 5min Resuspended the cell with 100μL chilled 0.1M CaCl2

Constructed strains with both PmerT-GFP(pSB3K3) and Pc(1-18I)-RBS-MerR(pSB1A2) Transformation the competent bacteria of PmerT-GFP(pSB3K3) with 2μL Pc(1-18I)-RBS-MerR(pSB1A2) plasmid.

7.19-7.25

Inducement of the strain with two plasmids which were constructed last week 7.20 With Hg(II)’s concentration = 1E-6 mol/L, 1E-7 mol/L and 1E-8 mol/L to certify that the strain worked. 7.22 With Hg(II)’s concentration = 1E-10, 1E-9, 1E-8, 2E-8, 4E-8, 6E-8, 8E-8, 1E-7, 1E-6, 1E-5(mol/L) 3 duplicates GFP’s intensity was measured by microplate reader. 7.23 With Hg(II)’s concentration = 1E-10, 1E-9, 1E-8, 4E-8, 7E-8, 1E-7, 4E-7, 7E-7, 1E-6, 1E-5(mol/L) 3 duplicates 7.24 With Hg(II)’s concentration = 1E-10, 1E-9, 1E-8, 4E-8, 7E-8, 1E-7, 4E-7, 7E-7, 1E-6, 1E-5(mol/L The OD was measured by Spectrophotometer to conform Hg(II)’s effect on bacterial growth

7.25 With Hg(II)’s concentration = 1E-10, 1E-9, 4E-9, 1E-8, 1.5E-8, 2E-8, 3E-8, 4E-8, 7E-8, 1E-7, 1.5E-7, 2E-7, 3E-7, 4E-7, 7E-7, 1E-6, 4E-6, 1E-5(mol/L) OD600 of each sample was also measured by Spectrophotometer.

7.26-8.2

Measurement of the 3 – dimensional figure which represented the time and dose response curve to mercury both for OD600 and GFP intensity.

I first tried to sample 100μL every 20min to the black-96-well plate and stored the plate in 4℃，OD was measured by spectrophotometer right after sampling.

The optimized protocol was to incubate the bacteria with Hg(II) in the microplate (black one for GFP intensity measurement and transparent one for OD600 measurement), then the two plates were measured by the microplate reader every 20min. During the interval of measurement, the two plates were cultured in the 37℃ shaker.

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