Difference between revisions of "Team:CGU Taiwan/standard content"

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Time: 40min<br>
 
Time: 40min<br>
 
Result:<br>
 
Result:<br>
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<img src="https://static.igem.org/mediawiki/2015/3/35/CGU-GPCR-note-0812-1.jpg" ><img src="https://static.igem.org/mediawiki/2015/9/90/CGU-GPCR-note-0812-2.jpg"><br>
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<div><img src="https://static.igem.org/mediawiki/2015/3/35/CGU-GPCR-note-0812-1.jpg" ><img src="https://static.igem.org/mediawiki/2015/9/90/CGU-GPCR-note-0812-2.jpg"></div><br>
 
Note: This gel result shows size difference of plasmid transformed or untransformed.<br>
 
Note: This gel result shows size difference of plasmid transformed or untransformed.<br>
 
<br>
 
<br>

Revision as of 17:06, 18 November 2015

Home | CGU_Taiwan

Home | CGU_Taiwan

Yeast With IL-8 ReceptorToehold Switches as RNA sensor

Lab Note


Yeast With IL-8 Receptor

2015.7.1

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Extraction of gDNA of Far1∆::KANMX strain
  2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
  3. PCR gDNA of Far1∆ ::KANMX
  4. Electrophoresis to check PCR product

Experiment steps:
< Extraction of gDNA of Far1∆::KANMX strain>
Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7


1. Design of primers
NamePur. Seq.(5’-3’) Size (mer.) MW (g/mol)Tm (℃)GC (%)Nmoleμl for 100μM
dFAR1 F’Desalt ggTTTTgTTAggCgggCAAg 20 6244.1 53.8 5521.25212.50
dFAR1 R’Desalt CATTAACTgCTATTTACgACgC 22 6669.4 51.1 4117.12171.20

2. PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 52℃ 30s
Step4→step2 for 30 cycle 72℃ 2min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

3.PCR reagent
10x Dream Taq buffer 2.5μl
2.5mM dNTP0.5μl
10mM primer(F)0.5μl
10mM primer(R)0.5μl
template (Far1∆::KANMX strain gDNA) 3.4μl
Taq polymerase0.5μl
ddH2O 17.1μl
Total volume25μl


< Electrophoresis to check PCR product>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample: 2μlPCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:
  M：Marker；#1：Far1 ∆ for annealing at 52℃
  There is no band appears in the gel electrophoresis.
Conclusion:
  Due to the Figure 1 in result, we have to check the temperature of primers annealing and the design of primers to solve the problem. Next, we use gradient PCR to find out the exactly temperature of primer annealing and we add positive control as well.

2015.7.2

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Extraction of gDNA of FAR1∆::KANMX strain
  2. Detection the concentration of fast extracted gDNA of FAR1∆::KANMX strain
  3. First round of PCR
  4. Electrophoresis to check first round-PCR product
  5. Second round of PCR

Experiment steps:
< Extraction of gDNA of FAR1∆::KANMX strain>
Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX 1.72 0.78 42.7
Positive control 1.63 0.76 37.1

1. Information of primers
NamePur. Seq.(5’-3’) Size (mer.) MW (g/mol)Tm (℃)GC (%)Nmoleμl for 100μM
dFAR1 F’Desalt ggTTTTgTTAggCgggCAAg 20 6244.1 53.8 5521.25212.50
dFAR1 R’Desalt CATTAACTgCTATTTACgACgC 22 6669.4 51.1 4117.12171.20

2.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 Gradient42℃-46℃-50℃ 30s
Step4→step2 for 30 cycle 72℃ 2min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

3.PCR reagent
10x Dream Taq buffer 2.5μl
2.5mM dNTP0.5μl
10mM primer(F)0.5μl
10mM primer(R)0.5μl
template (Far1∆::KANMX strain gDNA) 3.4μl
Taq polymerase0.5μl
ddH2O 17.1μl
Total volume25μl


< Electrophoresis to check first round-PCR product (25μl ul)>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample: 2μl PCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:
M: Marker; #1: FAR1∆ annealing at 42 ℃; #2: FAR1∆ annealing at 46 ℃;
#3: FAR1∆ annealing at 50 ℃; #4: FAR1∆ annealing at 52 ℃(positive control)

1.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 46℃30s
Step4→step2 for 30 cycle 72℃ 2min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr


2.PCR reagent
10x Dream Taq buffer 5μl
2.5mM dNTP1μl
10mM primer(F)1μl
10mM primer(R)1μl
template (First round PCR product) 1μl
Taq polymerase1μl
ddH2O 40μl
Total volume50μl

2015.7.3

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. 1.Electrophoresis to check second round-PCR product
  2. Transform PCR product into FUS::GFP(His) strain
Experiment steps:
< Electrophoresis to check second round-PCR product (50μl)>
Material:
  DNA marker: 100bp ladder 8μl   DNA sample:2μl PCR product + 1ul 6x loading buffer Condition: 0.5xTBE buffer 100V
Time: 30min

Result:
Figure 1. Gel electrophoresis of FAR1∆
M: Marker; #1: FAR1∆ annealing at 46 ℃

Consult the protocol < protocol of yeast transformation>
Use strain name: FUS1-GFP(His)
Selection plate: YPD+G418

2015.7.6

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Check transformation result
  2. Second round PCR
  3. Electrophoresis to check PCR product
  4. Incubate E.coli with shuttle vector-p426GAL1 from stock
Experiment steps:

1. Take plates out from incubator and observe growth of colony
2. Conclusion: We failed to transformation of PCR product so we should it again.


Consult the experiment record <2015.7.2 Experiment Record>

< Electrophoresis to check second round-PCR product>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample:2μlPCR product + 1μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result: Conclusion: Its expected length is 1.9kb and it worked.

< Incubate E.coli with shuttle vector-p426GAL1 from stock>
1. Take stock E.coli with shuttle vector-p426GAL1 from -80℃ fridge.
2. Plate E.coli on LB+Amp plates.
3. Incubate in 37℃ overnight.

2015.7.7

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Transform PCR product into FUS::GFP (His) strain
Experiment steps:

Consult the experiment record <2015.7.3 Experiment Record>

2015.7.9

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Miniprep plasmid of p426GAL1
  2. Measure concentration of plasmid.

Experiment steps:
< Miniprep plasmid of p426GAL1
Consult the protocol


  concentration 260/280 260/230
P426GAL1/1130.9ng/μl1.91 2.36
P426GAL1/2121.3ng/μl1.89 2.26

2015.7.12

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Second round PCR
  2. Incubate FAR1△::KANMX strain in 5ml YPD+A medium.
Experiment steps:

Consult the experiment record <2015.7.2 Experiment Record>

2015.7.17

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Transform PCR product into FUS1-GFP strain
Experiment steps:
< Transform PCR product into FUS1-GFP strain>
Consult the experiment record <2015.7.3 Experiment Record>

2015.7.20

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Maintain the colonies of far1Δ::KanMX-FUS1-GFP.
  2. 2nd round PCR for far1Δ::KanMX
  3. Phenol chloroform and EtOH precipitation of 2nd round PCR product
Experiment steps:
< Maintain the colonies of far1Δ::KanMX-FUS1-GFP >
  1. Choose 10 colonies to transfer the new YPD+G418 plate.
  2. Check plates after two days.

<2nd round PCR for far1Δ::KanMX
1.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 46℃30s
Step4→step2 for 30 cycle 72℃ 2min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

2.PCR reagent
10x Dream Taq buffer 5μl
2.5mM dNTP1μl
10mM primer(F)1μl
10mM primer(R)1μl
template (First round PCR product) 1μl
Taq polymerase1μl
ddH2O 40μl
Total volume50μl


< Phenol chloroform and EtOH precipitation of 2nd round PCR product >
Consult the protocol

2015.7.21

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Transform PCR product(Far1Δ::KanMX) into FUS1-GFP strain
  2. Extraction of gDNA of yeast
  3. Check PCR for far1Δ::KanMX-FUS1-GFP
  4. Digestion of p426 Gal
Experimental steps: < Transform PCR product into FUS1-GFP strain> Consult the experiment record <2015.7.3 Experiment Record> Because last time , negative control also had grown colony so we did transformation again. At the same time , we selected colony on selection plate to do PCR check.

< Extraction of gDNA of yeast>
Consult the protocol < protocol of fast extraction of gDNA of yeast>

< Check PCR for Far1Δ::KanMX- FUS-GFP >

1.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 46℃30s
Step4→step2 for 30 cycle 72℃ 90sec
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

2.PCR reagent
10x Dream Taq buffer 2.5μl
2.5mM dNTP0.5μl
10mM primer(F)0.5μl
10mM primer(R)0.5μl
template (First round PCR product) 8μl
Taq polymerase0.5μl
ddH2O 12.5μl
Total volume25μl
far1Δ::KanMX-FUS-GFP 260/280 260/230 ng/μl
1 1.76 0.91 111.7
2 2.05 1.32 158.2
3 2.02 1.29 120.4
4 1.98 1.08 89.9
5 2.05 0.95 66.9
6 2.07 1.45 182.6
7 1.86 0.90 95.4
8 2.08 1.45 166.4
9 2.18 0.42 22.5
10 2.07 1.30 102.4


< Digestion of p426 Gal >
  Eco RI(μl) Bam HI(μl) Uncut(μl)
ddH2O 20.5 20.5 21.5
10x NEB buf. #4 2.5 2.5 2.5
DNA(200ng) 1.5 1.5 1.5
Enzyme 0.5 0.5 --
total 25 25 25

1. Incubate at 37 for 1 hr.
2. Run the sample on the 1% agarose gel at 100 V for 30 min.(10 μl sample +2 μl loading dye)

2015.7.23

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Digestion of p426 Gal and rho-CXCR1
  2. Gel extraction of p426 Gal(vector)
  3. Clean up of rho-CXCR1(insert)
Experimental steps:
< Digestion of p426 Gal and rho-CXCR1>
  121 ng/μl 131 ng/μl
p426 Gal (2 μg) 16.5μl 15.2μl
10x NEB buf. 4 5μl 5μl
Bam HI 2μl 2μl
Eco RI 2μl 2μl
ddH2O 24.5μl 25.8μl
Total 50μl 50 μl

Rho-CXCR1 8(400 ng)
10x NEB buf. 4 5μl
Bam HI 1μl
Eco RI 1μl
ddH2O 35μl
Total 50μl

< Gel extraction of p426 Gal >
Consult the protocol < protocol of gel extraction>

< Clean up of rho-CXCR1>
Consult the protocol

2015.7.24

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Ligation of the rho-CXCR1 and p426 Gal
  2. Transform the ligation product into the E.coli
Experimental steps:
< Ligation of the rho-CXCR1 and p426 Gal>
  1:3 Vector only
Vector(11.1 ng/μl) 10μl 10μl
Insert(9.1 ng/μl) 7μl 0μl
Ligase 1μl 1μl
Ligation buff. 2μl 2μl
ddH2O 0μl 7μl
total 20μl 20μl
Incubate the ligation product at R.T. for 2 hr.

< Transform the ligation product into the competent cell>
Consult the protocol < protocol of ligation and transformation of E.coli>

2015.7.27

Operator: Wan-Yun, Jin-Ting
Goal:
  1. Fast extraction of gDNA of Far1Δ::KanMX-FUS1-GFP
  2. Check PCR for far1Δ::KanMX-FUS-GFP
Experimental steps: < Fast extraction of gDNA of far1Δ::KanMX-FUS-GFP > Consult the protocol
Strains 260/280 260/230 C(ng/μl)
FAR1∆::KANMX 1.85 1.19 177.3
FAR1∆::KANMX 1.83 1.19 163.8
Positive control 1.96 1.61 235.3

< Check PCR for Far1Δ::KanMX-FUS1-GFP>
1.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 46℃30s
Step4→step2 for 30 cycle 72℃ 90sec
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr


2.PCR reagent
  FAR1∆::KANMX Positive control
10x Dream Taq buffer 2.5μl2.5μl
2.5mM dNTP0.5μl0.5μl
10mM primer(F)0.5μl0.5μl
10mM primer(R)0.5μl 0.5μl
template (First round PCR product) 0.92μl0.64μl
Taq polymerase0.5μl0.5μl
ddH2O 19.58μl19.86μl
Total volume25μl25μl

2015.7.28

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1.Fast extraction of pGAL426 rho-CXCR1
Experimental steps:
< Fast extraction of plasmid DNA >
  1. Add 50 μl of STE buffer(100 mM NaCl, 10 mM Tris buffer, pH 7.0, 1 mM EDTA) into each new Eppendorf tube.
  2. Add 50 μl of Phenol chloroform and vortex vigorously for 30 sec.
  3. Centrifuge at 13,000 g for 5 min.
  4. Remove the 10 μl of supernatant to a new Eppendorf tube and run the sample on the 1 % agarose gel.

2015.7.29

Operator: Wan-Yun, Jin-Ting
Goal:
  1. Miniprep of p426 Gal-rho-CXCR1
  2. Enzyme digestion to check p426 Gal-rho-CXCR1
Experimental steps:
< Miniprep of p426 Gal-rho-CXCR1>
Consult the protocol

< Enzyme digestion for the p426 Gal-rho-CXCR1 check >
1. Enzyme digestion
p426 Gal-rho-CXCR1 #7,9 +,#8,12,16
10x NEB buff.4 2.5μl 2.5μl
Eco RI 0.5μl 0.5μl
Bam HI 0.5μl 0.5μl
DNA 2μl 1μl
ddH2O 19.5μl 20.5μl
total 25μl 25μl


2. Loading 10 μl sample and 2 μl DNA loading dye into the 1 % agarose gel.

2015.7.30

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Transform pSB1C3 BBa_J04450 into the competent cell
  2. Transform the plasmid (pRS405) into the competent cell
Experimental steps:
< Transform pSB1C3 BBa_J04450 into the competent cell>
Consult the protocol
Selection plate: LB+ Cam plate

< Transform the plasmids (pRS405) into the competent cell>
Selection plate: LB+ Amp plate

2015.8.3

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Miniprep of pRS405 and run the 1% agarose gel
  2. Digestion of pRS405 with Xho I, Bam HI and Eco RI
  3. Digestion of p426 Gal with Xho I
  4. Inoculate the bacteria with BBa_J04450 into the LB+ chloramphanicle broth


Experiment steps:
< Miniprep of pRS405 and run the 1% agarose gel >
Consult the protocol < protocol of miniprep plamid>

< Digestion of pRS405 with Xho I, Bam HI and Eco RI >
 uncut Xho I Bam HI Eco RI
pRS405 (V105○1) 0.5 0.5 0.5 0.5
10x NEB buff.4 2.5 2.5 2.5 2.5
Enzyme- 0.5 0.50.5
ddH2O22.5 21.5 21.5 21.5
total 25 μl 25 μl25 μl 25 μl
Reaction condition: 37℃, 1hr

< Digestion of p426 Gal with Xho I >
.uncut Xho I
p426 Gal (131 ng/μl)0.50.5
10x NEB buff.42.5 2.5
Enzyme- 0.5
ddH2O22.521.5
total25 μl 25 μl
Reaction condition: 37℃, 1hr

2015.8.4

Operator: Wan-Yun, Jin-Ting
Goal:
  1. 1st PCR of rho-CXCR1
  2. Electrophoresis to check the PCR product
  3. Miniprep of pSB1C3 BBa_J04450
Experiment steps:
<1st PCR of rho-CXCR1>
1. PCR program
Step Temp. Time
Step1 95℃ 5 min
Step2 95℃ 30 sec
Step340,44,48℃ 30 sec
Step4→Step2 for 30 cycle  72℃80sec
Step572℃ 5 min
Step6(Hold on) 10℃1 hr

2. PCR reagent
10x Dream Taq buffer 2.5μl
2.5mMdNTP  0.5μl
10μMprimer(F’) 0.5μl
10μMprimer(R’) 0.5 μl
Rho-CXCR13μl
Taq polymerase 0.5 μl
ddH2O17.5μl
Total volume25 μl

< Electrophoresis to check the PCR product>
< Electrophoresis to check second round-PCR product>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample:2μlPCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:

Conclusion: It expected length is 1200bp and it worked.
< Miniprep of pSB1C3 BBa_J04450>
Consult the protocol

2015.8.10

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Electrophoresis to check p426Gal-rho-CXCR1 digestion product again
  2. Do digestion of Lucy-rho-CXCR1 (insert) and p426Gal (vector)
  3. Clean up digestion product of Lucy-rho-CXCR1
  4. Gel extraction of digestion product of p426Gal
  5. Ligation insert and vector
  6. Transform the construct into competent cell
Experiment steps:
< Electrophoresis to check p426 Gal-rho-CXCR1 digestion product again>
Material:
  DNA marker: 1kb ladder 6ul
  DNA sample:2ul p426Gal-rho-CXCR1 digestion product(BamHI and XhoI) + 1ul 6x loading buffer
Condition:
   0.5xTBE buffer 100V
Time:
  30min Result:

Note: pGAL426 rho-CXCR1 enzyme digestion product with BamHI and XhoI
  Lucy-rho-CXCR1 (insert) p426Gal (vector) p426Gal (vector)
10x NEB buffer#4 5μl 5μl 5μl
BamHI 1μl 1μl 1μl
XhoI 1μl 1μl 1μl
DNA 25μl (400ng) 7μl (2ug) 7μl
ddH2O 18μl 34μl 34μl
Total volume 50μl 50μl 50μl
Reaction condition : 37℃ for 1hr
< Clean up digestion product of Lucy-rho-CXCR1>
Consult the protocol
Conc 260/280 260/230
10.0ng/ul 1.95 0.94

< Gel extraction of digestion product of p426Gal>
Consult the protocol
Conc. 260/280 260/230
11.2ng/ul 1.75 0.20

< Ligation insert and vector>
Vector:insert=1:3
  Test tube Vector only
Vector(11.2ng/ul) 9μl 9μl
Insert(10.0ng/ul) 6μl 0μl
Ligase 1μl 1μl
10xLigation buffer 2μl 2μl
ddH2O 2μl 8μl
Total volume 20μl 20μl
Reaction condition: Room temperature for 2-4hr

< Transform the construct into competent cell>
Selection plate: LB+Cam

2015.8.11

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Obeserve colony growth to confirm transformation result
  2. PCR Gpa1-Leu2
  3. Electrophoresis to check PCR product
  4. Second PCR round of Gpa1-Leu2 with first product
  5. Extraction of DNA with Phenol/Chloroform
Experiment steps:
< Obeserve colony growth to confirm transformation result>
Select 12 colonies and incubate in LB broth(with Amp) overnight,37℃

< PCR Gpa1-Leu2>
1.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 48℃,53℃,59℃,62℃ 90s
Step4→step2 for 30 cycle 72℃ 5min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

2.PCR reagent
10x Dream Taq buffer 2.5μl
2.5mM dNTP0.5μl
10mM primer(F)0.5μl
10mM primer(R)0.5μl
Template(pRS405,150ng) 0.55μl
Taq polymerase0.5μl
ddH2O 20μl
Total volume25μl


< Electrophoresis to check second round-PCR product (50μl)>
Material:
  DNA marker: 1kb ladder 6μl
  DNA sample:2μl PCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:

Conclution: Its expected length is 1502bp and it worked.

< Second PCR round of Gpa1-Leu2 with first product>
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 53℃ 30s
Step4→step2 for 30 cycle 72℃ 5min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr
2. PCR reagent
10x Dream Taq buffer 5μl
2.5mM dNTP1μl
10mM primer(F)1μl
10mM primer(R)1μl
template (First round PCR product) 1μl
Taq polymerase1μl
ddH2O 40μl
Total volume50μl

< Extraction of DNA with Phenol/Chloroform>
Consult the protocol

2015.08.12

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
     1. Miniprep of p426Gal-Lucy-rho-CXCR1
  2. Electrophoresis to confirm whether transformed successfully or not
  3. Digestion of p426Gal-Lucy-rho-CXCR1
  4. Electrophoresis to confirm which colony been transformed successlly
  5. Transform Gpa1 PCR product into FAR1::KANMX-FUS1-GFP strain
< Miniprep of p426Gal-Lucy-rho-CXCR1>
Consult the protocol

< Electrophoresis to confirm whether transformed successfully or not>
Material: DNA marker: 1kb ladder 6ul
DNA sample: plasmid 2ul + 3ul 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 40min
Result:

Note: This gel result shows size difference of plasmid transformed or untransformed.

< Digestion of p426Gal-Lucy-rho-CXCR1>
DNA(p426Gal-Lucy-rho-CXCR1 #2 #3 #7) 1μl
BamHI 0.5μl
XhoI >0.5μl
10x NEB buffer #4 2.5μl
ddH2O 20.5μl
Total volume 25μl

Condition: incubate at 37℃ for 1hr

< Electrophoresis to confirm which colony been transformed successlly>
Material: DNA marker: 1kb ladder 6μl
DNA sample: plasmid 5μl + 3μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:

Note: We use BamHI and XhoI to check whether the construct was built successfully.

< Transform Gpa1 PCR product into FAR1::KANMX-FUS1-GFP strain>
Consult the protocol
Yeast strain: FAR1::KANMX-FUS1-GFP
Selection plate : YPD+G418 plate

Toehold Switch As RNA Sensor

2015.6.20~2015.6.25

A, IL8 ; B, IL1β ; C, DUSP1 (dual specificity phosphatase 1) ;
D, SAT (spermidine/spermine N1-acetyltransferase EST


We use the website RNA structure to predict the secondary structure of these four toehold switches whether would have hairpin structures we want. Luckily, all of its results are match our expectation.

2015.6.25

Sent our designed sequences to IDT.

2015.7.1~2015.7.14

Amplify parts DNA we need.

2015.7.15

Receive IDT DNA.

2015.7.27

Transform BBa_I712019.

2015.7.28

Small scale incubation.

2015.7.29

Mini prep BBa_I712019 (Luciferase).
Concentration=186.2 (ng/ μl)

2015.7.30

Digest 4 toehold switches with EcoRI and XbaI and 4 trigger DNAs with XbaI and SpeI.
Plasmid DNA (25 ng/ μl)5 μl
NEB #21 μl
EcoRI (20 U/μl)0.2 μl
XbaI (20 U/μl0.2 μl
ddH2O3.6 μl
Total volume10 μl
Incubate at 37˚C for 3 hr.
Plasmid DNA ( 25 ng/ μl))5 μl
NEB #21 μl
EcoR I (20 U/μl)0.2 μl
SpeI (20 U/μl)0.2 μl
ddH2O3.6 μl
Total volume10 μl
Incubate at 37˚C for 3 hr.

2015.7.31

Do the electrophoresis of digested IDT products and gel extraction Restriction enzyme digestion of synthetic toehold and trigger RNA with EcoRI and SpeI. Samples were run in 1% agarose gel. Lane 1, 1kb DNA marker；Lane 2-5, 125 ng of synthetic DNA fragment that would transcribe into toehold sensors (SAT、DUSP1、IL-1β、IL-8 ) were digested by EcoRI and SpeI；Lane 6-9, 125 ng of DNA fragment that would transcribe into trigger RNAs (SAT、DUSP1、IL-1β、IL-8 ) were digested by EcoRI and SpeI.
All the results are around 100 bp, are the same as our expectation.

2015.8.3

Ask NYMU_Taiwan team for confirmed BBa_I712019, because we mistook the wrong antibiotic and lose all the parts DNA.

2015.8.4

Get the parts and transform parts BBa_I712019.
Vector DNA2 μl
Competent cell (DH10β)100 μl

2015.8.5

Do small scale incubation.

2015.8.6

MIniprep BBa_I712019.

2015.8.10

Small scale parts plasmids BBa_I712019 (Luciferase) digestion
Plasmid DNA (25 ng/ μl))2.5 μl
NEB #21 μl
EcoRI (20 U/μl)0.2 μl
SpeI (20 U/μl)0.2 μl
ddH2O6.1 μl
Total volume10 μl
Incubate at 37˚C for 3 hr.

2015.8.11

Do the electrophoresis of BBa_I712019 (Luciferase) to check the size and condition for experiment and gel extraction to check the size.

Mini-prep trigger RNAs in pSB1AK8 backbone (total 20 tubes).

2015.8.12

Largely cut luciferase in pSB1AK8 and purify it, then we got linear luciferase.
Plasmid DNA (142.5 ng/ μl)3 μl
Cut smart3 μl
XbaI (20 U/μl)0.6 μl
EcoRI (20 U/μl)0.6 μl
ddH2O22.8 μl
Total volume30 μl
Incubate at 37˚C for 3 hr.
Lane 1 and 4, 3 μg of luciferase digested by EcoRI and XbaI ; Lane 2, 1K DNA marker ; Lane 3, 3 μg of luciferase not digested by EcoRI and XbaI.In the picture, uncut plasmid is higher than the cut ones. After discussing with our advisor, we suppose that the uncut plasmid is not functional fold, so it runs slower than the plasmid with digestion.

Ligation toehold switches with luciferase (pSB1AK8).

Ligation trigger RNAs into pSB1AK8 backbone.

2015.8.13

Transform two kinds of construct into DH10α bacteria respectively to do mass culture.

2015.8.14

Sent our E. coli and primer (VF2) to sequence for check our assembled result.

2015.8.17

Have synthetic toeholds (DUSP1, SAT, IL-8) -luciferase in pSB1AK8 sequence be confirmed.

2015.8.18

Small scale T7 promoter digestion.

2015.8.19

Large scale T7 promoter digestion, and gel extraction.
Plasmid DNA30 μl
NEB #25 μl
PstI (20 U/μl)3 μl
SpeI (20 U/μl)2.6 μl
ddH2O10 μl
Total volume50 μl
Incubate at 37˚C for 3 hr.
Lane 1, marker ; Lane 2, 3 μg T7 promoter digested by restriction enzyme PstI and SpeI.

2015.8.20

Toehold+luciferase RNA enzyme digestion, gel extraction.
SAT1 241.7 (ng/ μl)
DUSP236.2 (ng/ μl)
IL8181.2(ng/ μl)

2015.8.21

Ligation trigger RNA to T7 promoter in pSB1AK8 backbone.
T7 promoter linear (119.6 ng/ μl) 15 μl
Plasmid DNA 2 μl
NEB #21.5 μl
PstI (20 U/μl)3 μl
SpeI (20 U/μl)3 μl
ddH2O5.5 μl
Total volume30 μl
The trigger RNA construct we finally complete. The structure contain T7 promoter, trigger, also Ampicillin antibiotic sequence easy for us to do the selection.

2015.8.22

Synthetic toehold–luciferase (IL1β) sequences confirmation.

T7 promoter-trigger RNA (SAT) in pSB1AK8 backbone sequences confirmation success, but T7 promoter-trigger RNA (DUSP1, IL1β,IL8) in pSB 1AK8 backbone sequences confirmation failed.

2015.8.23

Reconstruct the T7 promoter-trigger RNA (IL8,IL1 β,DUSP) in pSB1AK8 backbone.

2015.8.24

T7 promoter-trigger RNA(DUSP1, IL-1β,IL-8) in pSB1AK8 quick screen 6 colonies each.
1. Add100 μl lysis buffer into Eppendorf tube.
2. Use stick to take a little bit bacteria into lysis buffer.
3. Add 1:1 phenol/chloroform and shake it.
4. Centrifuge(12000g, 5mins).
5. Take 10μl to electrophoresis.

2015.8.25

Toehold switches digestion and ligation with pSB1C3 backbone.
Plasmid DNA (SAT1)45 μl
NEB#26 μl
EcoRI (20 U/μl)4 μl
PstI (20 U/μl)4 μl
ddH2O1 μl
Total volume60 μl


Plasmid DNA (DUSP1)45 μl
NEB#26 μl
EcoRI (20 U/μl4 μl
PstI (20 U/μl4 μl
ddH2O1 μl
Total volume60 μl


Plasmid DNA (IL8)45 μl
NEB#26 μl
EcoRI (20 U/μl4 μl
PstI (20 U/μl4 μl
ddH2O1 μl
Total volume60 μl


Plasmid DNA (IL1β)45 μl
NEB#26 μl
EcoRI (20 U/μl4 μl
PstI (20 U/μl4 μl
ddH2O1 μl
Total volume60 μl


BBa_I712074 linear5 μl
Toehold switch DNA4 μl
T4 ligase1 μl
PEG4002 μl
Total volume20 μl
The construct of toehold switches part we done. The structure include T7 promoter, toehold switches, and reporter gene—the luciferase. What’s more, the plasmids contain the chloramphenicol resistance gene for selection.

2015.8.26

Transformation
Vector DNA (158 ng/μl)1 μl
Competent cell (DH10β)100 μl

2015.8.27

Pick single colony and do the quick screen to check if the parts are inserted.

2015.8.28

Small scale incubation.

2015.8.31

Do the miniprep.
DNA Concentration= 174ng/μl

2015.9.1

Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp, CAM, Amp+CAM.

2015.9.2

T7-trigger (IL-1 β) ligation. In the figure, each lane are 10 μl of PCR products, we use temperature gradient (50°C, 53°C, 56°C, 59°C, 62°C, 65°C) to test the product of which temperature condition is suitable.
we choose the result of 53 °C for PCR. Because the product of 53 °C condition is much clearer, also much brighter then other conditions.

2015.9.3

Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp+CAM .
Toehold plasmid100 ng
Trigger plasmid100 ng
Competent cell (BL21DE3)100 μl

2015.9.6

1. Grow the overnight culture before the day of experiment.
2. Dilute 200 μl overnight culture with 10 ml LB broth and incubate for 2 hours.
3. Control the culture O.D between 0.6~0.8.
4. Spin down(6000g,3 mins) 9 ml culture in Eppendorf tube.
5. Wash the pellet with 1 ml PBS and spin down (6000 g, 3 min).
6. Add 500μl 1x luciferase passive lysis buffer and move to new mini beads beater tube with enough mini beads.
7. Put the tube into mini beads beater and shock 40 sec/time.
8. Shock 4~6 times and 2 mins on ice between every shock.
9. Spin down(12000g, 5mins)the lysate and move the supernatant into new Eppendorf tube.
10. Add 1ml 1X protein assay dye into cuvette and mix with 1μl lysate sample.
11. Put the sample into DU800 spectrometer and get the concentration data.
12. Normalize the quantity of protein(0.72 mg/ml) in each Eppendorf tube.
13. Add 50μl luciferin to each sample and put into GloMax® luminometer.
14. Get the number of luciferase activity.

2015.9.7

Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp+CAM again to have another plate of PCR T7-luciferase (positive).
Toehold plasmid100 ng
Trigger plasmid100 ng
Competent cell (BL21DE3)100 μl

2015.9.8

Luciferase assay

2015.9.9

Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp+CAM .

2015.9.10

Luciferase assay Failed!!! No luciferase express.

2015.9.11

Do the electrophoresis of negative control (T7 promoter with toehold switch).

2015.9.12

Miniprep to prepare the toehold switch IL1β.

Luciferase assay twice.

2015.9.13

Miniprep sensors and triggers in pSB1C3 (Il8,SAT1,DUSP1) and IL1β.
Luciferase assay twice.

2015.9.14

Luciferase assay once.

2015.9.16

Luciferase assay once

2015.9.17

Luciferase assay twice.