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 3.3 7/10 3.4 7/11 3.5 7/12 4 7/15~7/19 4.1 7/15 4.2 7/16 4.3 7/17 4.4 7/18 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

7/10

7/11

7/12

7/15~7/19

7/15

7/16

7/17

7/18

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