Contents 1 August 1.1 8/1 1.1.1 Inoculation & incubation 1.2 8/2 1.2.1 Check the following tubes: 1.2.2 Get plasmids: 1.3 8/5 1.3.1 Digestion: (biobrick part: restriction enzyme) for 2 hours 1.3.2 Ligtion(gene part-gene part) 1.3.3 Transform total 20 plates 1.3.4 PCR of original biobrick 1.3.5 Functional assay testing ~No results 1.4 8/6 1.4.1 Transformation results: 1.4.2 Original biobrick PCR results are similar to yesterday’s. 1.4.3 Functional assay test~ No results 1.5 8.7 1.5.1 Trouble shooting: Used the wrong primer for original biobrick PCR 1.5.2 Check results: 1.6 8.8 1.6.1 Sequencing results: 1.6.2 Plasmid extraction 1.7 8.9 1.7.1 Cloning =Digest+ligase+transform 1.7.2 PCR product has undergone blue-white selection, and will need to be incubated under Kanamycin and Ampicillin LB broth. 1.7.3 Functional assay 1.8 8.12 1.8.1 Digestion(gene part : restriction enzyme) 1.8.2 Ligation (some material were from past digestion products, which are already well cut) 1.8.3 Transformation: 6 plates (Ampicillin resistant) 1.8.4 Inoculation (5 tubes for each plate, total of 20 tubes) 1.9 8.13 1.9.1 Transform results(All grew, except BCI-pCI Cb grew too much ) 1.9.2 Inoculation/ Incubation(Ampicillin resistant, each plate inoculates 5 tubes, with a total of 25 tubes) 1.9.3 Check 1.9.4 Plasmid miniprep 1.9.5 Digestion (gene part: restriction enzyme) 1.9.6 Gel electrophoresis: pCI Cb is possible to fail because insert is nowhere seen 1.9.7 Ligation 1.9.8 Transformation (Ampicillin resistant) 1.10 8.14 1.10.1 Transform results: 1.10.2 Inoculation (Ampicillin resistant, 5 tubes each plus 1 tube of ACE) 1.10.3 Original biobrick 1.10.4 Check 1.10.5 Plasmid Miniprep 1.10.6 Digestion 1.10.7 Ligation 1.10.8 Transformation 1.11 8.15 1.11.1 Transform Result 1.11.2 Inoculation (Ampicillin resistant, 22 tubes total) 1.11.3 Original biobrick 1.11.4 Check 1.11.5 Plasmid miniprep 1.11.6 Digestion 1.11.7 Ligation 1.11.8 Transformation 1.12 8.16 1.12.1 Check inoculated bacteria 1.12.2 Transform results: 1.13 8.19 1.13.1 Plasmid miniprep(genepart-concenration) 1.13.2 Check ABC-Rhl l 1.13.3 Digestion 1.13.4 Ligation: some used previous digestion products 1.13.5 Transformation: total 5 plates(Ampicillin resistant) 1.13.6 Inoculation(Ampicillin resistant, 24 tubes total) 1.14 8.20 1.14.1 Check: 1.14.2 Plasmid miniprep: 1.14.3 Transform(1 plate) 1.14.4 Inoculation:(Ampicillin resistant) 1.14.5 Sequencing: sending 10 samples(R=reverse primer, F=forward primer) 1.15 8.22 1.15.1 Plasmid miniprep 1.15.2 Digest(gene part-restriction enzyme) 1.15.3 Ligation & Transform: 1.15.4 Inoculation:

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

Get plasmids:

gene	name	conc(ng/ul)
Pc-RBS-cinR-tt	Pc Acin-2	144.3
	Pc Acin-5	142.6
Pcin-RBS-cinR-RBS-GFPmut-tt	Bcin Cg-1	182.9
	Bcin Cg-2	173.9
	Bcin Cg-3	120
	Bcin Cg-4	129.7
Pcin-RBS-cinR-RBS-mCherryt-tt	Bcin Cm-1	104.4
	Bcin Cm-2	105.4
	Bcin Cm-3	103.2
	Bcin Cm-4	118.5
RBS-BFP-tt	Cb-1	186.8
	Cb-2	191.9
	Cb-3	214.3
RBS-Luciferase-tt	Cl-1	266.4
	Cl-2	249.1
RBS-CI-tt	RBS-CI-tt-1	159.3
	RBS-CI-tt-2	166.1

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.

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)

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:

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

Gene	Name	size	conc
pCI-RBS-GFP-tt	pCI E1 2	916	81.5
	pCI E1 4		74.1
pCI-RBS-BFP-tt	pCI Cb 2	898	97.4
	pCI Cb 4		110
pCI-RBS-mCherry-tt	pCI B5 2	907	102.1
	pCI B5 3		101.3
pCI-RBS-luciferase-tt	pCI Cl 4	1129	85.4
	pCI Cl 5		146.6
RBS-LasR	RBS lasR 1	867	128.7
	RBS lasR 2		125.7
Pc-RBS-RhlR-tt-pRhl-RBS-RhlR	PcA C1 3	1705	93
	PcA C1 4		74.5
Pc-RBS-CinR-tt-pCin-RBS-CinR	PcAcin Bcin 1993		wrong sequence
RBS-LuxR	RBS luxR 1 796 69.1
	RBS luxR 2	101.4
Pc-RBS-CinR-tt-pCin-RBS-CinR-RBS-
GFP-tt	ABC cing	2860
Pc-RBS-CinR-tt-pCin-RBS-CinR-RBS
-mCherry-tt	ABC cinm	2851
pCin-RBS-CinR-RBS-BFP-tt	Bcin Cb 1	1876	118.2
	Bcin Cb 2		113
pCin-RBS-CinR-RBS-Luciferas-tt	Bcin Cl 1	2110	141.3
	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

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

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

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

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

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

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

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:

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:

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