Contents 1 July 2 7/1~7/5 2.1 7/1 2.1.1 Transform: 2.2 7/2 2.2.1 Result: 2.2.2 Transform: 2.2.3 Inoculation and incubation at LB broth 2.3 7/3 2.3.1 Inoculation and incubation at LB broth 2.3.2 Check 2.4 7/4 2.4.1 Check and Plasmid DNA extraction (mini-prep): 2.5 7/5 2.5.1 Check: (for plasmid ready to be sequenced) 3 7/8~7/12 3.1 7/8 3.1.1 Sequence: 3.1.2 Primer list: 3.1.3 Digestion, ligation, transform: 3A assembly and standard assembly both 3.2 7/9 3.2.1 Result: 3.2.2 Inoculation and incubation: 3.3 7/10 3.3.1 Plasmid DNA extraction (mini-prep): 3.3.2 Result: 3.3.3 Re-inoculation and incubation of plate of B0030-CinR 3.4 7/11 3.5 =Plasmid DNA extraction (mini-prep): 3.5.1 Result: 3.6 7/12 3.6.1 Digestion: standard assembly only: 3.6.2 Result: 4 7/15~7/19 4.1 7/15 4.1.1 Ligation: 4.1.2 Transform: 4.2 7/16 4.2.1 Result: 4.2.2 Conclusion: 4.3 7/17 4.3.1 Digestion and ligation: 4.3.2 Transform: 4.3.3 Sequence: 4.3.4 Sequence results: 4.4 7/18 4.4.1 Inoculation and Incubation at LB broth: 4.5 7/19 5 7/22~7/31 5.1 7/22 5.2 7/23 5.3 7/24 5.4 7/25 5.5 7/26 5.6 7/27 5.7 7/28 5.8 7/29 5.9 7/30 5.10 7/31 5.11 July 6 2

July

7/1~7/5

Finish RhlR and LasR circuit.

7/1

Transform:

1. Positive feedback circuit:
   Pc-A-C-B, Pc-A-C-E, Pc-A-C-B, Pc-D-F-B, Pc-D-F-E, Pc-D-F-G;
2. Control:
   PLas-B, PLas-E, PLas-G, PRhl-B, PRhl-E, PRhl-G;
(A: RBS-RhlR-tt C: PRhl-RBS-RhlR D: RBS-LasR-tt F: PLas-RBS-LasR B: RBS-mCherry-tt E: RBS-GFPmut-tt G: RBS-mRFP-tt )
3. New:
   Pcin, CinR (resistant=C)

7/2

Result:

There’s no colonies at plates of Pcin and CinR.

Transform:

Pcin(resistant=A), CinR (resistant=K)

Inoculation and incubation at LB broth

1. Positive feedback circuit:
Pc-A-C-B, Pc-A-C-E, Pc-A-C-B, Pc-D-F-B, Pc-D-F-E, Pc-D-F-G;
2. Control:
PLas-B, PLas-E, PLas-G, PRhl-B, PRhl-E, PRhl-G; (A: RBS-RhlR-tt C: PRhl-RBS-RhlR D: RBS-LasR-tt F: PLas-RBS-LasR B: RBS-mCherry-tt E: RBS-GFPmut-tt G: RBS-mRFP-tt )

7/3

Inoculation and incubation at LB broth

Pcin(resistant=A), CinR (resistant=K)

Check

1. Positive feedback circuit:
Pc-A-C-B, Pc-A-C-E, Pc-A-C-B, Pc-D-F-B, Pc-D-F-E, Pc-D-F-G;
2. Control:
PLas-B, PLas-E, PLas-G, PRhl-B, PRhl-E, PRhl-G; (A: RBS-RhlR-tt C: PRhl-RBS-RhlR D: RBS-LasR-tt F: PLas-RBS-LasR B: RBS-mCherry-tt E: RBS-GFPmut-tt G: RBS-mRFP-tt )

7/4

Check and Plasmid DNA extraction (mini-prep):

1. Positive feedback circuit:
Pc-A-C-B, Pc-A-C-E, Pc-A-C-B, Pc-D-F-B, Pc-D-F-E, Pc-D-F-G;
2. Control:
PLas-B, PLas-E, PLas-G, PRhl-B, PRhl-E, PRhl-G; (A: RBS-RhlR-tt C: PRhl-RBS-RhlR D: RBS-LasR-tt F: PLas-RBS-LasR B: RBS-mCherry-tt E: RBS-GFPmut-tt G: RBS-mRFP-tt )
3. Pcin(resistant=A), CinR (resistant=K)

7/5

Check: (for plasmid ready to be sequenced)

ACB-4, ACE-5, ACG-1, DFB-1, DFE-2, DFE-3, DFE-4, DFE-5, DFG-1, RhlB-1, RhlE-2, RhlG-1, RhlG-2, LasB3, LasE1, LasG4 (8,9,11 well failed :DFE-4/DFE-5/ RhlB-1)

7/8~7/12

Construct the circuit for new receptor CinR

7/8

Sequence:

ACB-4, ACE-5, ACG-1, DFB-1, DFE-2, DFE-3, DFG-1, RhlE-2, RhlG-1, RhlG-2, LasB3, LasE1, LasG4

Primer list:

Primer order	Name	sequence
BMRC-120	iGEM2013-pSB1A2	attaccgcctttgagtgagc	R
BMRC-153	iGEM2013-pSB1A2	gtgccacctgacgtctaagaa	F
BMRC-122	iGEM2013-mCherry	gccgtcctcgaagttcatcac	mcherry
BMRC-124	iGEM2013-mRFP	aacggtaacaccaccgtc	RFP
BMRC-126	iGEM2013-GFP	cttgtagttcccgtcatcttt	GFP

Digestion, ligation, transform: 3A assembly and standard assembly both

B0030+CinR

7/9

Result:

The plate of B0030+CinR following 3A assembly was OK. The other following standard assembly failed unexpectedly.

Inoculation and incubation:

B0030+cinR

7/10

Plasmid DNA extraction (mini-prep):

B0030-cinR

Result:

A260/A280=1.3

Re-inoculation and incubation of plate of B0030-CinR

7/11

=Plasmid DNA extraction (mini-prep):

B0030-cinR

Result:

A260/A280=1.3

7/12

Digestion: standard assembly only:

B0030 CinR

Result:

There’s no band for B0030

7/15~7/19

CinR, LuxR

7/15

Ligation:

B0030+CinR (The certain content in the ependorf labeled B0030 undergone digestion was actually previously digested; thus, concentration was assumed to be low)

Transform:

B0030-CinR pCI CI

7/16

Result:

Only one colony of plate(B0030-CinR) grew. CI are successfully incubated. pCI failed to grow.

Conclusion:

B0030 threw away.

7/17

Digestion and ligation:

B0030 (eppendorf from early stage)

Transform:

B0030-CinR

RBS(B0030)

pCI(R0051)

LuxR(C0062)

pLux(Lux pR, R0062)

pConst(J23119)

mTagBFP(K592100)

Luciferase(J52008)

simple Las detecting system(K575024)

simple Rhl detecting system(K575033)

Sequence:

PcDFE, PcDFG, Pc ACB, Rhl B, Rhl E Total 16 tube

Sequence results:

No single plasmid was correct at all.

7/18

Inoculation and Incubation at LB broth:

B0030-CinR, B0030, LuxR, pLux, pConst(J23119), mTagBFP, Luciferase, simple Las detecting system(K575024), simple Rhl detecting system(K575033)

7/19

7/22~7/31

7/22

7/23

7/24

7/25

7/26

7/27

7/28

7/29

7/30

7/31

July

2