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 4.5.1 Check and Plasmid DNA extraction (mini-prep): 5 7/22~7/31 5.1 7/22 5.1.1 Digestion: 5.1.2 Ligation overnight at 16 degree incubator: 5.1.3 Transform: 5.1.4 Primer design and Sequence: 5.2 7/23 5.2.1 Results: 5.2.2 Transform: 5.2.3 Original biobrick: 5.2.4 Human practice: 5.3 7/24 5.3.1 Result: 5.3.2 Ligation at room temperature for 3 hours: 5.3.3 Transformation (correct competent cell – DH5α) 5.3.4 Original biobrick: 5.4 7/25 5.4.1 Results 5.4.2 Transformation 5.4.3 Inoculation and Incubation: 5.4.4 Original biobrick: 5.5 7/26 5.5.1 Results: 5.5.2 Official biobricks 5.5.3 Transformation 5.5.4 Inoculation and Incubation 5.5.5 Make new Amp plates 5.6 7/29 5.6.1 Check: 5.6.2 Inoculation and Incubation at LB broth: 5.7 7/30 5.7.1 Check: 5.7.2 Digestion, Ligation and Transformation: 5.8 7/31 5.8.1 Digestion, Ligation and Transformation: 5.8.2 Plasmid DNA Extraction (mini-prep):

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

Check and Plasmid DNA extraction (mini-prep):

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

7/22~7/31

Construct our circuit.

7/22

Digestion:

B0030, CI, mTagBFP, Luciferase, B0015(tt), pCin, ACin (RBS-CinR), BCin (RBS-CinR), B0030 (RBS), LuxR

Note:Because the gel for electrophoresis healed since we took away the comb too early, most of the digestion products overflew from the wells. And we almost couldn’t see the band after dyeing, so we redid the digestion step. The second time we finally got our material for ligation! But LuxR had very strange bands (too large), so we decided not use it. And retransformed it from official plasmid.

Ligation overnight at 16 degree incubator:

(1) B0030+CI

(2) B0030+mTagBFP

(3) B0030+Luciferase

(4) B0030-CinR+tt

(5) pCin+B0030-CinR

Transform:

(1) pCI (Amp, Chl): to check the drug resistance was correct.

(2) LuR (Chl)

(3) pCin (Amp)

Primer design and Sequence:

Design primers for AbaR, pqsR.

Start checking sequencing results.

7/23

Results:

Transformation: All 4 plates didn’t grow

Transform:

(1) B0030-CI (Amp)

(2) B0030-mTagBFP (Amp)

(3) B0030-Luciferase (Amp)

(4) [B0030-CinR]-tt (Amp)

(5) pCin-[B0030-CinR] (Amp)

(6)(7) pCI (Amp, Chl)

(8) pCin (Amp)

(9) LuxR (Chl) x2

Original biobrick:

(1) Got P.aeruginosa & A.baumannii (on blood agar plate)

(2) Each liquid culture x3 tubes

Human practice:

Visited the department of laboratory medicine in NTUH

7/24

Result:

(1) Transformation: all 10 plates didn’t growth…

(2) Debug: We took the wrong competent cell(HB101)…

(3) Sequencing: for functional assay, only the positive feedback of RhlR-GFP (ACE) was all correct. Others need to be redone.

Ligation at room temperature for 3 hours:

(1) B0030-CI

(2) B0030-mTagBFP

(3) B0030-Luciferase

(4) [B0030-CinR]-tt

(5) pCin-[B0030-CinR]

Transformation (correct competent cell – DH5α)

(1) B0030-CI (Amp)

(2) B0030-mTagBFP (Amp)

(3) B0030-Luciferase (Amp)

(4) [B0030-CinR]-tt (Amp)

(5) pCin-[B0030-CinR] (Amp)

(6)(7) pCI (Amp, Chl)

(8) pCin (Amp)

(9) LuxR (Amp): we took the wrong LuxR previously, but this time it was correct.

(10) B0015 (tt) (Amp)

Original biobrick:

(1) Extracted the whole genome of P. aeruginosa

7/25

Results

(1) Transformation: 7 growth except pCI and LuxR

Transformation

(1) PcA (pConst18C-B0030-RhlR-tt)

(2) PcD (pConst18C-B0030-LasR-tt)

(3) G5 (B0030-mRFP-tt)

(4) E1 (B0030-GFP-tt)

(5) B5 (B0030-mCherry-tt)

(6) C1 (pRhl-B0030-RhlR)

(7) F2 (pLas-B0030-LasR)

(8) pCI

(9) LuxR

No.1~7 are going to make stocks.

Inoculation and Incubation:

(No.1~5 cultured 5 tubes, 6 & 7 cultured 2 tubes, total 29 tubes)

(1) B0030-CI (Amp)

(2) B0030-mTagBFP (Amp)

(3) B0030-Luciferase (Amp)

(4) B0030-CinR-tt (Amp)

(5) pCin-B0030-CinR (Amp)

(6) pCin (Amp)

(7) B0015 (Amp)

Original biobrick:

(1) Extracted the whole genome of A. baumannii

(2) The concentration wasn’t very high

7/26

Results:

(1) Inoculation: forgot to put in 37 degree incubator…(put in 4 degree refrigerator)

(2) Transformation: 7 growth except pCI and LuxR…

(3) Debug: purified LuxR official plasmid, but the concentration was very~low

Official biobricks

(1) Take pCI and LuxR from 2012 official kit (at Life Science college)

Transformation

(1) pCI (Amp)

(2) LuxR (Amp)

(3) ACE (positive feedback of RhlR-GFP) (Amp): for functional assay

Transform at room temperature over weekend.

Inoculation and Incubation

(1) Keep inoculating at 37 degree incubator (since they didn’t growth very well)

(2) But the quality is uncertain

Make new Amp plates

7/29

Check:

(1)B0030-CI (Amp)

(2)B0030-mTagBFP (Amp)

(3)B0030-Luciferase (Amp)

(4)B0030-CinR-tt (Amp)

(5)pCin-B0030-CinR (Amp)

(6)pCin (Amp)

(7)B0015 (Amp)

B0015 contained a backbone of 3K. Check again!?

Inoculation and Incubation at LB broth:

(1)PcA (pConst18C-B0030-RhlR-tt)

(2)PcD (pConst18C-B0030-LasR-tt)

(3)G5 (B0030-mRFP-tt)

(4)E1 (B0030-GFP-tt)

(5)B5 (B0030-mCherry-tt)

(6)C1 (pRhl-B0030-RhlR)

(7)F2 (pLas-B0030-LasR)

(8)pCI

(9)LuxR

(10)ACE

7/30

Check:

pCI-1, ACE, LuxR, C1, PcA, E1-1, C1, B5, PcD, F2, G5

Digestion, Ligation and Transformation:

Pc+AcinR

BcinR+C GFPmut(E1)

BcinR +C mCherry(B5)

B0030-mTagBFP+B0015

B0030-Luci+B0015

B0030-CI+B0015

7/31

Digestion, Ligation and Transformation:

Pc+AcinR

BcinR+CGFPmut(E1)

B0030-mTagBFP+B0015

B0030-Luci+B0015

B0030-CI+B0015

pLas + RBS-LasR failed two times (without bands at gel extracting)

11 plate Pc+AcinR 30 / 31

BcinR+CGFPmut(E1) 30 / 31

BcinR +CmCherry(B5) 30

B0030-mTagBFP+B0015 30/31

B0030-Luci+B0015 30/31

B0030-CI+B0015 30/31

Plasmid DNA Extraction (mini-prep):

gene	name	vector	size(bp)	conc(ng/ul)	site
pCI-1	Bba_R0051	pSB1A3	49	136	yellow box
pCI-2	Bba_R0051	pSB1A4	49	66.6	yellow box
LuxR-1	Bba_C0062	pSB1A2	781	119	yellow box
LuxR-3	Bba_C0062	pSB1A2	781	119.8	yellow box
RhlR-GFPmut3-positive	ACE-2	pSB1A2	2572	308.8	yellow box
	ACE-4	pSB1A2	2572	287.9	yellow box
Pc-RBS-Rhl-tt	PcA-1	pSB1A2	908	82.3	yellow box
	PcA-3	pSB1A2	908	79.8	yellow box
Pc-RBS-Las-tt	PcD-2	pSB1A2	902	72.4	yellow box
	PcD-3	pSB1A2	902	81.5	yellow box
pLas-RBS-LasR	seguence is not correct
pRhl-RBS-RhlR	C1-2	pSB1A2	797	104.2	yellow box
	C1-3	pSB1A2	797	119.2	yellow box
RBS-GFPmut3-tt	E1-1	pSB1AK3	867	129.2	yellow box
	E1-3	pSB1AK3	867	91.1	yellow box
RBS-mCherry-tt	B5-1	pSB1AK3	858	105.1	yellow box
	B5-3	pSB1AK3	858	73.2	yellow box
RBS-mRFP-tt	seguence is not correct