Team:NTU-Taida/Notebook/Journal/August

From 2013.igem.org

(Difference between revisions)
(→Sequencing results:)
Line 198: Line 198:
==8.9==
==8.9==
===Cloning =Digest+ligase+transform===
===Cloning =Digest+ligase+transform===
-
#Pc(18C) / Acin Pc A Cin
+
#Pc(18C) / Acin   Pc A Cin
#Pc(18A) / Acin  Pc A Cin
#Pc(18A) / Acin  Pc A Cin
#pCinR / RBS-CinR  B Cin
#pCinR / RBS-CinR  B Cin
Line 205: Line 205:
#RBS-LuxR 1 / tt  A Lux
#RBS-LuxR 1 / tt  A Lux
#PcA C1-3 / Cg,Cm,Cb,Cl  ABC Rhl g,m,b,l
#PcA C1-3 / Cg,Cm,Cb,Cl  ABC Rhl g,m,b,l
-
#PcA / C1 PcA C1(or PcA rhl)
+
#PcA / C1  PcA C1(or PcA rhl)
Because some bands are irregular, these bands fail to be cloned.
Because some bands are irregular, these bands fail to be cloned.

Revision as of 20:37, 27 September 2013

Journal

August
Sun. Mon. Tue. Wed. Thu. Fri. Sat.
01 02 03
04 05 06 07 08 09 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

Contents

August

8/1

Inoculation & incubation

  1. Pc+AcinR (5 tubes)
  2. BcinR+CGFPmut(E1) (5 tubes)
  3. B0030-mTagBFP+B0015 (5 tubes)
  4. B0030-Luci+B0015 (5 tubes)
  5. B0030-CI+B0015 (5 tubes)

Total 30 tubes

8/2

Check the following tubes:

  1. PcAcin2,5
  2. BcinCg1-5
  3. BcinCm1-5
  4. Cb1-5
  5. Cl 1-5
  6. RBS-CI-tt 1-5
NTU-Taida-journal-August-1.jpg
NTU-Taida-journal-August-2.jpg

Get plasmids:

genenameconc(ng/ul)
Pc-RBS-cinR-ttPc Acin-2144.3
 Pc Acin-5142.6
Pcin-RBS-cinR-RBS-GFPmut-ttBcin Cg-1182.9
 Bcin Cg-2173.9
 Bcin Cg-3120
 Bcin Cg-4129.7
Pcin-RBS-cinR-RBS-mCherryt-ttBcin Cm-1104.4
 Bcin Cm-2105.4
 Bcin Cm-3103.2
 Bcin Cm-4118.5
RBS-BFP-ttCb-1186.8
 Cb-2191.9
 Cb-3214.3
RBS-Luciferase-ttCl-1266.4
 Cl-2249.1
RBS-CI-ttRBS-CI-tt-1159.3
 RBS-CI-tt-2166.1

8/5

Digestion: (biobrick part: restriction enzyme) for 2 hours

  1. pCI: S/P
  2. E1(B0030-GFPmut3-tt): X/P
  3. B5(B0030-mCherry-tt): X/P
  4. Cb3(B0030-mTagBFP-tt): X/P
  5. Cl1(B0030-luciferase-tt): X/P
  6. PcA(pConst-B0030-RhlR-tt): S/P
  7. C1(pRhl-B0030-RhlR): X/P
  8. B0030: S/P
  9. LasR: X/P
  10. LuxR: X/P
  11. PcACin2 & 5:S/P
  12. BCin3:X/P, S/P
  13. BCinCm(pCin-RBS-CinR-RBS-mCherry-tt): X/P
  14. BCinCg(pCin-RBS-CinR-RBS-GFP-tt): X/P

Ligtion(gene part-gene part)

  1. pCI-E1
  2. pCI-B5
  3. pCI-Cb
  4. pCI-Cl
  5. B0030-LasR
  6. B0030-LuxR
  7. PcA-C1
  8. PcACin-BCin x2
  9. PcACin-BCinCm x2
  10. PcACin-BCinCg x2
  11. BCin-Cb
  12. BCin-Cl

Transform total 20 plates

Ampicillin resistant plates (total of 15 plates)

  1. pCI-E1
  2. pCI-B5
  3. pCI-Cb
  4. pCI-Cl
  5. B0030-LasR
  6. B0030-LuxR
  7. PcA-C1
  8. PcACin-BCin x2
  9. PcACin-BCinCm x2
  10. PcACin-BCinCg x2
  11. BCin-Cb
  12. BCin-Cl

Chloramphenicol resistant plates (total of 5 plates)

  1. K575033
  2. K575024
  3. I13522
  4. I13521
  5. S03335

PCR of original biobrick

Band length is strange. Needs further PCR.

NTU-Taida-journal-August-3.jpg

Functional assay testing ~No results

8/6

Transformation results:

 All plates grew. Inoculation of the followings: (5 tubes each, 2 tubes fail, with a total of 78 tubes)
  1. pCI-E1
  2. pCI-B5
  3. pCI-Cb
  4. pCI-Cl
  5. B0030-LasR
  6. B0030-LuxR
  7. PcA-C1
  8. PcACin-BCin x2
  9. PcACin-BCinCm x2
  10. PcACin-BCinCg x2
  11. BCin-Cb
  12. BCin-Cl
  13. K575033
  14. K575024
  15. I13522
  16. I13521
  17. S03335

Original biobrick PCR results are similar to yesterday’s.

Functional assay test~ No results

8.7

Trouble shooting: Used the wrong primer for original biobrick PCR

 New PCR results (annealing temperature at 52∘C)
NTU-Taida-journal-August-4.jpg

Further process:

  1. PCR with rTaq polymerase: add 1 A to terminals
  2. Ligation the three tubes overnight (plasmid undergo blue-white selection)

Check results:

NTU-Taida-journal-August-5.jpg
NTU-Taida-journal-August-6.jpg
NTU-Taida-journal-August-7.jpg

8.8

Sequencing results:

IV. 130806102

  1. Pc Acin-5 F
  2. Bcin Cg-1 F
  3. Bcin Cg-1 R
  4. Bcin Cg-3 F
  5. Bcin Cg-3 R
  6. Bcin Cm-1 F
  7. Bcin Cm-1 R
  8. Bcin Cm-2 F
  9. Bcin Cm-2 R
  10. Cb-1 F
  11. Cb-1 R
  12. Cl-1
  13. RBS-CI-tt

Results: PcAcin/ Bcin are wrong

Plasmid extraction

GeneNamesize conc
pCI-RBS-GFP-ttpCI E1 291681.5
 pCI E1 4 74.1
pCI-RBS-BFP-ttpCI Cb 289897.4
 pCI Cb 4 110
pCI-RBS-mCherry-ttpCI B5 2907102.1
 pCI B5 3 101.3
pCI-RBS-luciferase-ttpCI Cl 4112985.4
 pCI Cl 5 146.6
RBS-LasRRBS lasR 1 867128.7
 RBS lasR 2 125.7
Pc-RBS-RhlR-tt-pRhl-RBS-RhlRPcA C1 3170593
 PcA C1 4 74.5
Pc-RBS-CinR-tt-pCin-RBS-CinRPcAcin Bcin 1993 wrong sequence
RBS-LuxRRBS luxR 1 796 69.1  
 RBS luxR 2101.4 
Pc-RBS-CinR-tt-pCin-RBS-CinR-RBS-   
GFP-ttABC cing2860 
Pc-RBS-CinR-tt-pCin-RBS-CinR-RBS   
-mCherry-ttABC cinm2851 
pCin-RBS-CinR-RBS-BFP-ttBcin Cb 11876118.2
 Bcin Cb 2 113
pCin-RBS-CinR-RBS-Luciferas-ttBcin Cl 12110141.3
 Bcin Cl 2 87.1
K575033  214.6
K575024  331.9
I13522  236.6
I13521  380
S03355  274.5

8.9

Cloning =Digest+ligase+transform

  1. Pc(18C) / Acin Pc A Cin
  2. Pc(18A) / Acin  Pc A Cin
  3. pCinR / RBS-CinR  B Cin
  4. pLuxR / RBS-LuxR 2  B Lux
  5. pLasR /RBS-LasR 1  B Las
  6. RBS-LuxR 1 / tt  A Lux
  7. PcA C1-3 / Cg,Cm,Cb,Cl  ABC Rhl g,m,b,l
  8. PcA / C1  PcA C1(or PcA rhl)

Because some bands are irregular, these bands fail to be cloned.

PCR product has undergone blue-white selection, and will need to be incubated under Kanamycin and Ampicillin LB broth.

Functional assay

Run the ELISA plate reader with the control group which keeps expressing GFP. Still no successful results yet.

8.12

Digestion(gene part : restriction enzyme)

  1. B0030-CI-tt(=BCI) 2: S/P
  2. pCI-E1(GFP) 2: X/P
  3. pCI-B5(mCherry) 3: X/P
  4. pCI-Cb(BFP) 4: X/P
  5. pCI-Cl(luci) 5: X/P
  6. LuxR: X/P

Ligation (some material were from past digestion products, which are already well cut)

  1. BCI-pCI E1
  2. BCI-pCI B5
  3. BCI-pCI Cb
  4. BCI-pCI Cl
  5. B0030-LuxR
  6. pCin-[B0030-CinR]

Transformation: 6 plates (Ampicillin resistant)

  1. BCI-pCI E1
  2. BCI-pCI B5
  3. BCI-pCI Cb
  4. BCI-pCI Cl
  5. B0030-LuxR
  6. pCin-[B0030-CinR]

Inoculation (5 tubes for each plate, total of 20 tubes)

  1. Pc-ACin (18C) Amp resistant
  2. Pc-ACin (18A) Amp resistant
  3. BLas(pLas-B0030-LasR) Amp resistant
  4. PcA-C1 Amp resistant

8.13

Transform results(All grew, except BCI-pCI Cb grew too much )

  1. BCI-pCI E1
  2. BCI-pCI B5
  3. BCI-pCI Cb (grow too much, lack single colony)
  4. BCI-pCI Cl
  5. B0030-LuxR
  6. pCin-[B0030-CinR]

Inoculation/ Incubation(Ampicillin resistant, each plate inoculates 5 tubes, with a total of 25 tubes)

  1. BCI-pCI E1
  2. BCI-pCI B5
  3. BCI-pCI Cl
  4. B0030-LuxR
  5. pCin-[B0030-CinR] (=BCin)

Check

NTU-Taida-journal-August-8.jpg
NTU-Taida-journal-August-9.jpg 
 PcAcin bands are strange!

Plasmid miniprep

  1. BLas 1,2
  2. PcACin 18C 1,2
  3. PcACin 18A 2,5
  4. PcAC1 1,2

Digestion (gene part: restriction enzyme)

  1. pLas: S/P
  2. pRhl: S/P
  3. pCin 2: S/P
  4. B5 (mCherry): X/P
  5. pCI Cb: X/P

Gel electrophoresis: pCI Cb is possible to fail because insert is nowhere seen

Ligation

  1. pLas-Cb
  2. pRhl-B5
  3. pCin-E1
  4. pCI-Cb (redid it again)
  5. BCI-pCI B5
  6. BCI(vector only): to see whether digestion was clean enough.

Transformation (Ampicillin resistant)

  1. pLas-Cb
  2. pRhl-B5
  3. pCin-E1
  4. pCI-Cb
  5. BCI-pCI B5
  6. BCI

8.14

Transform results:

  1. pCI-Cb didn’t growth
  2. BCI (vector only) growth a little, prove that digestion was successful

Inoculation (Ampicillin resistant, 5 tubes each plus 1 tube of ACE)

  1. pLas-Cb
  2. pRhl-B5
  3. pCin-E1
  4. BCI-pCI B5
  5. ACE : for stock

Original biobrick

Inoculation 20 tubes (plate 163,134 – promoter PQS) Ampicillin resistant

Check

NTU-Taida-journal-August-10.jpg

BCI-pCI Cb looked failed, because inserts of BCIs all looked too short?

NTU-Taida-journal-August-11.jpg

RBS-LuxR had strange length~

Plasmid Miniprep

  1. BCI-pCI E1 1,2
  2. BCI-pCI Cl 4,5
  3. BCin 3,4

Digestion

  1. Pc18C (J23100): S/P
  2. Pc (J23119): S/P
  3. RBS-CinR: E/S
  4. PcAC1 1: S/P
  5. PcD: S/P
  6. BLas 3: X/P 
 →RBS-CinR didn’t had insert. Probably failed?

Ligation

  1. J23119(Pc18A)-ACin
  2. PcAC1-B5
  3. PcAC1-Cb
  4. PcAC1-Cl
  5. PcD-BLas

Transformation

  1. Pc18A-ACin Amp
  2. PcAC1-B5(ABC Rhl m) Amp
  3. PcAC1-Cb(ABC Rhl b) Amp
  4. PcAC1-Cl(ABC Rhl l) Amp
  5. PcD-BLas Amp
  6. LuxR Amp: from official gel stab

8.15

Transform Result

  1. All plates grew~
  2. Pc(18A)ACin growth(but only a little)! Yet its resistance was Chl…
  3. ABC Rhl m growth too much.

Inoculation (Ampicillin resistant, 22 tubes total)

  1. ABC Rhl b
  2. ABC Rhl l
  3. PcD-BLas
  4. LuxR
  5. Pc(18A)-ACin: check resistance, 2 tubes

Original biobrick

  1. Inoculation 20 tubes (plate 159,130 - pqsR) Amp
  2. Check result (Ppqs)

Check

NTU-Taida-journal-August-12.jpg
NTU-Taida-journal-August-13.jpg 
BCI-pCI B5 looked too short

Plasmid miniprep

  1. pCin E1 1,4
  2. BCI-pCI B5 2,3
  3. pLas-Cb 2,4
  4. pRhl 1,5

Digestion

  1. pCI 1: S/P
  2. Cb 1: X/P
  3. Cb 2: X/P
  4. Cb 3: X/P

Three Cb looked alike

Ligation

  1. pCI-Cb1
  2. pCI-Cb2
  3. pCI-Cb3
  4. Pc(18A)-ACin

Transformation

  1. pCI-Cb1 Amp
  2. pCI-Cb2 Amp
  3. pCI-Cb3 Amp
  4. Pc(18A)-ACin Chl
  5. ABC Rhl m Amp: transfer to another plate

Total 5 plates

8.16

Check inoculated bacteria

NTU-Taida-journal-August-14.jpg

1: PcD Blas4 2: PcD Blas5 3: PcD Blas3 4: PcD Blas1

5: LuxR 1 6: LuxR 2 7: LuxR 3 8: LuxR 4

9: LuxR 5 10: Rhlb2 11: Rhlb5 12: PcAcin try1

13: PcAcin try2 14: Rhlb1 15: Rhlb2

NTU-Taida-journal-August-15.jpg

1: Rhl b4 2: PQSR1 3: PQSR2 4: PQSR3

5: PQSR5 6: PQSR8 7: PQSR10 8: PQSR11

9: PQSR13 10: PQSR14 11: PQSR15 12: PQSR16

13: PQSR18 14: PQSR20

Transform results:

  1. ABC Rhlm(0)
  2. pCI-Cb1(0)
  3. pCI-Cb3(0)
  4. pCI-Cb2(0)
  5. Chl-PcACin(18A) (X)

8.19

Plasmid miniprep(genepart-concenration)

PcAC1-Cl 2 170.5 PcAC1-Cl 4 202.9 PcAC1-Cb 1 137.5 PcAC1-Cb 3 140 PcD-Blas 1 148 PcD-Blas 3 159.2 PcD-Blas 4 152.9 PQSR10 373.6 PQSR11 347.4 LuxR2 89.8 LuxR3 101.1

Check ABC-Rhl l

NTU-Taida-journal-August-16.jpg

The bands seemed to be weird. It should be around 2.8kb instead of 2k, therefore needs further sequencing to identify.

Digestion

  1. RBS-CinR: E/S
  2. pLas Cb#: X/P
  3. pRhl B5#: X/P
  4. BCI#: X/P
  5. RBS-LasR#: E/S→used wrong enzyme
  6. RBS-LasR#: E/S→used wrong enzyme
  7. pPQS(19): E/P
  8. linear pSB1C3: E/P→No results, probably too low concentration

Ligation: some used previous digestion products

  1. [RBS-CinR]-tt (=ACin)
  2. pLas-[RBS-LasR] (=BLas)
  3. PcD-pLas Cb (=OLasCb)
  4. PcA-pRhl B5 (=ORhlB5)
  5. PcAC1-BCI
  6. RBS-LasR

Transformation: total 5 plates(Ampicillin resistant)

  1. ACin
  2. BLas
  3. OLasCb
  4. ORhlB5
  5. PcAC1-BCI
  6. RBS-LasR

Inoculation(Ampicillin resistant, 24 tubes total)

  1. B0015: 5 tubes
  2. B0030: 5 tubes
  3. ABC Rhlm: 5 tubes
  4. pCI Cb###: each 3 tubes

8.20

Check:

NTU-Taida-journal-August-17.jpg

B0015 did not grow at all

Plasmid miniprep:

  1. B0030-2
  2. B0030-3
  3. ABC Rhl m-1
  4. ABC Rhl m-2
  5. pCI Cb2-1
  6. pCI Cb2-2

Transform(1 plate)

pSB1C3 backbone and promoter PQS are transformed after ligation.

Inoculation:(Ampicillin resistant)

  1. ACin Amp(5 tubes)
  2. BLas Amp(5 tubes)
  3. PcAC1-BCI Amp(5 tubes)
  4. RBS-LasR Amp(5 tubes)
  5. B0015 Amp(5 tubes)

Sequencing: sending 10 samples(R=reverse primer, F=forward primer)

  1. BCin# R
  2. pCI B5 F
  3. pCI E1 F
  4. pCI Cl F
  5. pCin E1 F
  6. pRhl B5 F
  7. ABC Rhl l F
  8. ABC Rhl l R
  9. ABC Rhl b F
  10. ABC Rhl b R

8.22

Check:

NTU-Taida-journal-August-18.jpg
NTU-Taida-journal-August-19.jpg

Plasmid miniprep

  1. B0015-2,3
  2. BLas-2,3
  3. RBS-LasR-3,4
  4. pCAC1-BCI-2,3
  5. Acin 1

Digest(gene part-restriction enzyme)

  1. pPQS19 E/S
  2. E1-1 E/X
  3. B5-1 E/X
  4. Cb3 E/X
  5. Cl1 E/X
  6. PcDBLas 3 S/P

Ligation & Transform:

  1. pPQS- E1 Amp
  2. pPQS- B5 Amp
  3. pPQS-Cb Amp
  4. pPQS-Cl Amp

Inoculation:

  1. pPQS+pSC1C3 backbone-5 tubes Chloramphenical resistant
  2. AbaR (on T vector)-5 tubes Ampicillin+Kanamycin resistant



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