Team:LZU-China/chz notes

Team:LZU 2015

Journal

Parts Construction

April
4.7-4.11

Primer designing
Designing primer of ribB:
Sense: 5-GGAATTC CGC TCTAGA ATGAATCAGACG-3;
Anti: 5-TTCTGCAG TTGG ACTAGT TCAGCTGGCT-3;

4.12-4.30

ribB PCR
Had operated PCR with genome DNA or bacterial liquid directly as template.

Template is	Genome DNA	Bacterial Liquid
Template volume	1μL	1μL
Primer FW	1μL	1μL
Primer F	1μL	1μL
Primer R	1μL	1μL
dNTP	1μL	1μL
Buffer	2μL	2μL
Taq enzyme	0.5μL	0.5μL
ddH₂O	13.5μL	13.5μL

May

ribB PCR

June

Since the results of PCR are not good enough, we decided to synthesis the CDS of ribB instead. The synthesized ribB CDS arrived in June 1st, connecting in pUC-19-Amp.

6.1-6.7

Parts K1755001, K1755002 construction

BBa_K1755001 construction

6.1

1. Extracted pSB1C3;

2. Coated and selected single colonies of bacteria containing pUC-19-Amp on Amp resistance;

3. selective medium, and activated them in LB liquid medium.

6.2

1. Extracted pUC-19-Amp;

2. Restriction enzyme digestion on pUC-19-Amp to obtain the DNA of ribB CDS.

Digestion System	50μL
Xba 1	1.5μL
Pst 1	1.5μL
10*M　buffer	5μL
pUC-19-Amp	15μL
H₂O	27μL

Afterwards run the electrophoresis of the product of the digestion, and extracted the target DNA by Gel Extraction Kit.

6.3

1. Performed Restriction enzyme digestion on pSB1C3;

Digestion System	50μL
Xba 1	1.5μL
Pst 1	1.5μL
10*M　buffer	5μL
pSB1C3	15μL
H₂O	27μL

Afterwards run the electrophoresis of the product of the digestion, and extracted the target DNA by Gel Extraction Kit.

2. Connected ribB CDS into pSB1C3. Thus we had constructed BBa_K1755001;

Connecting system	50μL
T4 DNA ligase	2.5μL
T4 Buffer	5μL
ribB CDS	10μL
pSB1C3	2.5μL
H₂O	30μL

3. Digested the pSB1C3-ribB CDS; Electrophoresis of the product of the digestion showed we had cut down the target DNA successfully:

Figure-1 ribB (K1755001) in pSB1C3 (digested by EcoRI). The marker is TAKARATM 1kb Ladder Marker (2015-6-3)

4. Transformed the pSB1C3-ribB CDS into DH 5α; then coated on chloramphenicol resistance selective solid medium.

BBa_K1755002 construction

6.4

1. Selected single colonies of bacteria containing pSB1C3;

1. 2. Activated in LB liquid medium under 37 centigrade;

3. Had stored the DH 5αwith pSB1C3-ribB CDS in 50% glycerol under -20℃; Extracted pSB1C3-ribB CDS;

4. Digested the pSB1C3-ribB CDS and the standard part BBa_K823017;

Digestion System	50μL
Spe 1	1.5μL
Pst 1	1.5μL
10*H　buffer	5μL
pSB1C3	15μL
H₂O	27μL

Digestion System	50μL
Xba 1	1.5μL
Pst 1	1.5μL
10*M　buffer	5μL
BBa_K823017	15μL
H₂O	27μL

Afterwards run the electrophoresis of the product of the digestion, and extracted the target DNA by Gel Extraction Kit.

5.Connected BBa_K823017 into the pSB1C3-ribB CDS. Thus we had constructed BBa_K1755002;

Connecting system	50μL
T4 DNA ligase	2.5μL
T4 Buffer	5μL
BBa_K823017	10μL
pSB1C3	2.5μL
H₂O	30μL

6. Digested the pSB1C3-BBa_K1755002; Electrophoresis of the product of the digestion showed we had cut down the target DNA successfully:

Figure-2 K1755002 in pSB1C3 (Di-digest, EcoRI and PstI ). TAKARATM 1kb Ladder Marker (2015-6-5)

7. Transformed the pSB1C3-BBa_1755002 into DH 5α; then coated on chloramphenicol resistance selective solid medium.

8. Activated in LB liquid medium under 37 centigrade;

9. Had stored the DH 5αwith pSB1C3-BBa_1755002 in 50% glycerol under -20℃; Extracted pSB1C3-BBa_K1755002;

6.8-6.14

Parts K1755004, K1755005, K1755006, K1755007, K1755008, K1755009, K1755305, K1755024 construction

6.8

Digested the pSB1C3-BBa_K1755002;

Digestion System	50μL
Xba 1	1.5μL
Pst 1	1.5μL
10*M　buffer	5μL
pSB1C3-BBa_K1755002	15μL
H₂O	27μL

Afterwards run the electrophoresis of the product of the digestion, and extracted the BBa_K1755002 by Gel Extraction Kit.

6.9-6.13

1.Digested all the standard parts needed;

Digestion System	50μL
Spe 1	1.5μL
Pst 1	1.5μL
10*H　buffer	5μL
pSB1C3-part	15μL
H₂O	27μL

Parts needed include: BBa_K525998, BBa_K608003, BBa_K608006, BBa_K608004, BBa_K608007, BBa_K911009;

Afterwards run the electrophoresis of the product of the digestion, and extracted the plasmids by Gel Extraction Kit.

BBa_K1755009, BBa_K1755024.

3. Digested the pSB1C3-New built parts; Here is some of the electrophoresis of the product

of the digestion that showed we had cut down the target DNA successfully:

Figure-3 K1755004 (Left 2 rows)，K1755305 (Middle 2 rows) and K1755009 (Right 2 rows).

The parts are all in pSB1C3 and was digested by EcoRI and PstI. TAKARATM 150bp Ladder

Marker (2015-6-9)

Figure-4 K1755006 (left row) and K1755024 (middle row). The parts were both digested by

EcoRI and PstI. TAKARATM 150bp Ladder Marker (2015-6-13)

6.14-6.16

6.17-6.23

Part K1755003 construction

July 7.27-7.29

Part K1755301 construction

The synthesized BBa_K1755301 had arrived in 7.13 which is packed in pUC-19-Amp.

7.27

Activated in LB liquid medium under 37 centigrade;

7.28

1. Extracted pUC-19-Amp;

2. Digested the pUc-19-Amp-BBa_K1755301 and pSB1C3;

Digestion System	50μL
EcoR 1	1.5μL
Pst 1	1.5μL
10*H Buffer	5μL
pUC-19-Amp/pSB1C3	15μL
H2O	27μL

Afterwards run the electrophoresis of the product of the digestion, and extracted

BBa_K1755301 and pSB1C3 by Gel Extraction Kit.

Connected pSB1C3 and BBa_K1755301.

Connecting system	50μL
T4 DNA ligase	2.5μL
T4 Buffer	5μL
BBa_K1755301	10μL
pSB1C3	2.5μL
H2O	30μL

August

Part K1755302 303 construction

Parts Examination

June

6.10-6.15

Parts K1755007 examination

6.10

Activated DH5αand DH-5αwith BBa_K1755007 in LB liquid medium under 37 centigrade;

6.11-6.13

1. Transferred 10mL bacterial liquid into 90mL MM medium;

2. Cultured in 37℃, shaking;

3. Examined the concentration of bacteria and riboflavin from the beginning, and each 12h

once.

OD600
	0h	12h	20h	36h	48h
DH 5α(1)	0.121	0.508	0.469	0.402	0.365
DH 5α(2)	0.141	0.530	0.487	0.427	0.415
K1755007(1)	0.204	0.501	0.711	0.664	0.643
K1755007(2)	0.174	0.487	0.713	0.653	0.629

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	12h	20h	36h	48h
DH 5α(1)	-0.112	0.001	0.009	0.004	-0.001
DH 5α(2)	-0.103	-0.001	0.015	0.015	0.001
K1755007(1)	-0.105	0.000	0.006	0.000	0.005
K1755007(2)	-0.103	-0.001	0.002	0.009	0.629

6.24-6.30

Parts K1755005 006 009 examination (first round)

6.24

Activated DH5α, DH-5αwith BBa_K1755005, BBa_K1755006, BBa_K1755009 in LB liquid

medium under 37 centigrade;

6.24-6.28

1. Transferred 10mL bacterial liquid into 90mL MM medium;

2. Cultured in 37℃, shaking;

3. Examined the concentration of bacteria and riboflavin from the beginning, and each 12h

once.

OD600
	0h	12h	20h	36h	48h	60h
DH 5α(1)	0.059	0.921	0.820	0.905	0.894	0.889
DH 5α(2)	0.066	0.927	0.921	1.001	0.989	0.937
K1755007(1)	0.068	0.959	0.976	1.012	1.008	0.974
K1755007(2)	0.063	0.856	0.831	0.908	0.905	0.909

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	12h	20h	36h	48h	60h
DH 5α(1)	0.013	0.005	0.076	0.009	0.013	0.009
DH 5α(2)	0.009	0.013	0.016	0.008	0.014	0.007
K1755007(1)	0.009	0.011	0.010	0.104	0.018	0.014
K1755007(2)	0.020	0.047	0.021	0.012	0.016	0.016

July

7,1-7,7

Parts K1755005 006 009 examination (second round)

7.1

Activated;

7.2-7.6

1. Transferred 1mL bacterial liquid into 100mL M9 (added 0.5% yeast) medium;

2. Cultured in 37℃, shaking;

3. Examined the concentration of bacteria and riboflavin from the beginning, and each 24h

once.

OD600
	0h	18h	41h	66h
DH 5α	0.018	1.499	1.520	1.501
K1755005	0.009	1.516	1.497	1.546
K1755006	0.011	1.4549	1.488	1.454
K1755009	0.009	1.470	1.435	1.470

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	18h	41h	66h
DH 5α	-0.011	0.000	-0.002	-0.001
K1755005	-0.003	0.002	0.003	0.009
K1755006	-0.011	-0.003	-0.002	-0.001
K1755009	-0.010	-0.002	-0.001	0.000

7.9-7.14

Parts K1755005 006 009 examination (third round)

7.9

Activated;

7.9-7.14

1. Transferred 1mL bacterial liquid into 100mL M9 (added 0.5% yeast) medium;

2. Cultured in 37℃, shaking;

3. Examined the concentration of bacteria and riboflavin from the beginning, and each 24h

once;

OD600
	0h	28h	48h
DH 5α	0.042	1.502	1.493
K1755005	0.059	1.576	1.576
K1755006	0.072	1.544	1.589
K1755009	0.052	1.420	1.650
DK1755004 10	0.039	1.295	1.304
K1755004 11	0.047	1.312	1.478
K1755004 12	0.050	1.294	1.447

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	28h	48h
DH-5α	0.005	0.037	0.029
K1755005	0.009	0.021	0.007
K1755006	0.011	0.009	0.008
K1755009	0.015	0.047	0.037
K1755004 10	0.016	0.024	0.037
K1755004 11	0.003	0.045	0.026
K1755004 12	0.004	0.038	0.039

7.16-7.26

Parts K1755005 006 009 examination (fourth round)

7.16-7.18

1. Activated;

2. Extracted the plasmids;

3. Transformed the plasmids into BL-21 and stored in 50% glycerol under -20℃;

7.19

Activated

7.20-7.24

1. Transferred 1mL bacterial liquid into 100mL M9 (added 1% casein hydrolysate) medium;

2. Covered the conical flasks with aluminized paper, cultured in 37℃, shaking;

3. Examined the concentration of bacteria and riboflavin from the beginning, and each 24h once;

OD600
	0h	24h	48h	72h	96h
K1755005	0.025	1.129	1.131	1.247	1.234
K1755006	0.021	1.134	1.170	1.212	1.259
K1755009	0.016	1.116	1.157	1.327	1.359
K1755004 12	0.031	1.294	1.359	1.374	1.460

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	24h	48h	72h	96h
K1755005	0.000	0.0045	0.0133	0.095	0.077
K1755006	-0.001	0.026	0.0127	0.054	0.058
K1755009	0.001	0.031	0.315	0.078	0.076
K1755004 12	-0.002	0.012	0.052	0.076	0.025

Figure-5 From left to right: blank M9 medium, BBa_K1755005, BBa_K1755006, BBa_K1755009, BBa_K1755004(12).

August

8.1-8.7

Part K1755301 examination (first round)

8.1-8.2

Transformed into BL-21 and stored in 50% glycerol under -20℃;
Activated;

8.2-8.6

1. Transferred 1mL bacterial liquid into 100mL M9 (added 1% casein hydrolysate) medium, added Cu²⁺ into the medium to 2mg/L, 5mg/L, 10mg/L, 15mg/L, 20mg/L;

2. Covered the conical flasks with aluminized paper, cultured in 37℃, shaking;

3. Examined the concentration of bacteria and riboflavin from the beginning, and each 24h once;

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	14h	24h	37h	48h	61h
BL-21	0.009	0.049	0.096	0.072	0.11	0.0115
0mg/L	-0.004	0.042	0.085	0.101	0.105	0.122
2mg/L	0.013	0.109	0.174	0.189	0.199	0.192
5mg/L	0.001	0.157	0.236	0.274	0.274	0.285
10mg/L	0.002	0.135	0.142	0.114	0.11	0.09
15mg/L	0.003	0.121	0.052	0.062	0.07	0.054
20mg/L	0.000	0.089	0.098	0.02	0.024	0.017

8.8-8.10

Part K1755301 examination (second round)

Familiar to the first round, but change the gradient of metal ion;
Cu²⁺: 0mg/L, 0.2mg/L, 0.4mg/L, 0.6mg/L, 0.8mg/L, 1mg/L, 2mg/L;

OD444 (1mLsupernatant + 2mL 0.1M HCl)

Cu²⁺	0h	12h	24h
BL-21	-0.006	0.052	0.142
0mg/L	0.024	0.043	0.108
0.2mg/L	0.021	0.017	0.058
0.4mg/L	-0.004	0.022	0.054
0.6mg/L	-0.005	0.036	0.098
0.6mg/L	-0.005	0.036	0.098
0.8mg/L	0.007	0.03	0.052
1mg/L	0.02	0.024	0.109
2mg/L	0.023	0.112	0.186

8.11-8.14

Part K1755301 examination (third round)

8.13-8.16

Parts K1755024 305 examination

Part BBa_K1755024 examination

F : 0, 20μM, 40μM, 60μM, 80μM

OD600

0h

6h

12h

20h

32h

56h

0

0.006

0.577

0.75

0.766

0.763

0.83

20μM

0.006

0.002

0.621

0.642

0.722

0.757

0.745

0.612

0.785

0.788

0.836

0.883

40μM

0.013

0.027

0.529

0.526

0.9

0.7

0.747

0.733

0.756

0.761

0.863

0.805

60μM

0.01

0.004

0.602

0.611

0.796

0.799

0.784

0.796

0.718

0.96

0.903

0.999

80μM

0.006

0.581

0.572

0.705

0.751

0.676

0.721

0.715

0.716

0.779

0.908

OD444 (1mLsupernatant + 2mL 0.1M HCl)

0h

6h

12h

20h

32h

56h

0

0.041

0.017

-0.02

0.06

0.057

0.056

20μM

0.022

0.014

0.018

0.01

0.021

0.0

0.086

0.038

0.056

0.072

0.041

0.043

40μM

0.016

0.049

0.009

0.011

-0.027

0.006

0.067

0.003

0.04

0.031

0.014

0.003

60μM

0.014

0.009

0.000

0.002

0.025

0.019

0.053

0.01

0.025

0.018

80μM

0.014

0.012

0.000

0.009

0.034

0.001

0.023

0.026

0.015

0.000

0.018

Part BBa_K1755305 examination

Hg²⁺: 0, 1μM, 5μM, 25μM, 100μM;

OD600
	0h	12h	24h	36h
BL-21	1.492	1.201	1.389	1.469
0	1.21 1.256	1.2 1.205	1.39 1.496	1.55 1.545
1μM	-0.011 -0.033 0.001	0.864 0.916 0.820	1.292 1.336 1.216	1.583 1.443 1.454
5μM	-0.002 -0.005 -0.005	0.832 0.253 0.243	1.369 1.303 1.469	1.530 1.552 1.557
25μM	-0.003 -0.003 -0.003	0.092 0.048 0.134	1.366 1.525 1.263	1.578 1.594 1.575
100μM	0.012 -0.001 -0.003	0.066 0.041 0.055	0.025 0.015 0.053	0.044 1.434 0.028

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	12h	24h	36h
BL-21	0.052	0.021	0.022	0.016
0	0.012 0.018	0.033 0.036	0.019 0.056	0.02 0.025
1μM	0.013 -0.006 0.005	0.014 0.031 0.029	0.009 0.007 0.001	0.006 -0.050 0.039
5μM	0.002 0.004 0.001	0.023 0.017 0.007	0.007 -0.004 -0.011	-0.004 -0.008 0.026
25μM	0.007 0.005 0.006	0.009 0.008 0.023	0.011 -0.012 -0.004	0.002 -0.010 0.017
100μM	0.029 0.003 0.004	0.015 0.02 0.025	0.009 0.001 0.004	0.002 0.001 0.003

8.26-8.29

Part K1755302 examination

Cu²⁺: 0, 1mg/L, 2mg/L, 3mg/L, 4mg/L;

OD600
	10h	22h	34h	46h	58h
0	0.680	0.801	0.963	1.103	1.058
1mg/L	0.656 0.654	0.772 0.874	0.889 1.080	1.048 1.112	1.044 1.027
2mg/L	0.669 0.689	0.821 0.855	0.832 0.850	0.954 0.981	1.071 0.933
3mg/L	0.577 0.523	0.715 0.527	0.723 0.592	0.728 0.696	0.654 0.669
4mg/L	0.452 0.449	0.469 0.402	0.460 0.429	0.735 0.652	0.822 0.871

OD444 (1mLsupernatant + 2mL 0.1M HCl)

10h

22h

34h

46h

58h

1mg/L

0.028

0.023

0.034

0.027

0.031

0.030

0.054

0.047

0.171

0.183

2mg/L

0.012

0.015

0.040

0.031

0.060

0.048

0.066

0.068

0.159

0.090

3mg/L

0.017

0.026

0.060

0.059

0.057

0.072

0.095

0.175

0.161

0.162

4mg/L

0.027

0.022

0.059

0.055

0.067

0.070

0.093

0.098

0.123

0.138

September

9.2-9.11

RT-PCR to examine the response of BBa_K1755301 to Cr6+

9.3

Wash the tips by DEPC

9.4

1.Activated the bacteria(BL-21, BL-21-BBa_K1755301), the dispose them to different

concentration of Cu2+ and Cr6+;

	Mental ion	Concentration
1	ddH2O
2	Cr6+	0.1mM
3	Cr6+	0.1mM
4	Cu2+	10mg/L
5	Cu2+	1mg/L

2.Extracted RNA, performed reverse transcription;

9.8

Performed PCR to test the primers.

Primer of ribB:

Sense：5-GGCCAGGACGATTCAGATCT-3

Anti：5-CTGAAGGTGTGACTACCGGT-3

Primer of 16s RNA:

Sense：5-CGATCCCTAGCTGGTCTGAG-3

Anti：5-CAATATTCCCCACTGCTGCC-3

9.11

Running Device

9.11

MFC construction (firsct round)

8.25

Sterilized all the component of MFC;

Prepare M9 medium and electrolyte;

8.26

Constructed the MFC

8.26-8.28

MFC testing (first round)

Examined the concentration of bacteria and riboflavin;

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	12h	24h	36h	48h
0	0.210	0.546	0.555	0.566	0.577
2mg/L	0.395	0.742	0.735	0.756	0.715
5mg/L	0.388	0.610	0.408	0.276	0.239
10mg/L	0.322	0.416	0.427	0.211	0.123

OD600
	0h	12h	24h	36h	48h
0	0.011	0.009	0.047	0.020	0.026
2mg/L	0.022	0.023	0.031	0.036	0.038
5mg/L	0.024	0.041	0.054	0.029	0.029
10mg/L	0.024	0.046	0.050	0.043	0.045

8.28-8.29

MFC construction (second round)

Similiar as before

MFC testing (second round)

OD600
	0h	12h	24h	36h	48h
0	0.472	0.337	0.113	0.145	0.207
0.5mg/L	0.455	0.093	0.347	0.349	0.379
1mg/L	0.422	0.076	0.339	0.353	0.391
1.5mg/L	0.449	0.102	0.181	0.236	0.298
2mg/L	0.441	0.292	0.122	0.140	0.239
3mg/L	0.463	0.119	0.118	0.208	0.303

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	12h	24h	36h	48h
0	0.019	0.029	0.027	0.021	0.025
0.5mg/L	0.019	0.027	0.027	0.033	0.035
1mg/L	0.007	0.028	0.012	0.018	0.036
1.5mg/L	0.034	0.052	0.030	0.034	0.039
2mg/L	0.020	0.098	0.046	0.043	0.038
3mg/L	0.040	0.090	0.050	0.043	0.051

9.01-9.02

MFC construction (third round)

Similar as before

9.02-9.04

MFC testing (third round)

OD600
	0h	12h	24h	36h	48h
0	0.376	0.073	0.281	0.126	0.227
1mg/L	0.404	0.087	0.351	0.379	0.391
2mg/L	0.396	0.080	0.176	0.167	0.254
3mg/L	0.392	0.082	0.181	0.212	0.322
4mg/L	0.407	0.093	0.194	0.178	0.147
5mg/L	0.406	0.180	0.130	0.134	0.134

OD444 (1mLsupernatant + 2mL 0.1M HCl)

	0h	12h	24h	36h	48h
0	0.017	0.024	0.017	0.024	0.023
1mg/L	0.022	0.030	0.024	0.034	0.029
2mg/L	0.014	0.029	0.014	0.029	0.022
3mg/L	0.034	0.041	0.038	0.039	0.034
4mg/L	0.029	0.072	0.028	0.040	0.047
5mg/L	0.030	0.076	0.066	0.051	0.048

Interlab

August

8.17-8.27

FCM

8.17

Interlab Device1 and 2 constructed.

8.22

Interlab Device3 constructed

9.2

Interlab data obtained and processed.

Protocol

Parts Construction

Plasmid Extraction

We used OMEGATM Plasmid Mini Prep Kit (D6943-02) to extracted plasmids.

1. Add 2mL bacteria liquid into a 2mL centrifuge tube;

2. Centrifuge the bacteria liquid at 12000r/min for 2 minutes;

3. Discard the upper clear liquid;

4. Add 250μL Solution I into the centrifuge tube. Vortex the tube until there is no solid

sediment left in the bottom;

5. Add 250μL Solution II into the centrifuge tube. Rotate the tube slightly until the liquid

being clear;

6. Add 350μL Solution III into the centrifuge tube. Rotate the tube several times until white

sediment appearing;

7. Centrifuge at 12000r/min for 20 minutes;

8. Transfer the upper liquid into a spin column with a collection tube, and centrifuge them at

10000r/min for 1 minute;

9. Discard the filtered liquid in the collection tube, then add 500μL HB Buffer into the spin

column. Centrifuge them at 10000r/min for 1 minute;

10. Discard the filtered liquid in the collection tube, then add 700μL DNA Wash Buffer into

the spin column. Centrifuge them at 10000r/min for 1 minute;

11. Perform step 10 again.

12. Discard the collection tube. Put the spin column standing for 1 or 2 minutes.

13. Transfer the spin column into a clean centrifuge tube. Add 30-50μL Elution Buffer which

has been preheated to 65℃, then centrifuge them at 10000r/min for 1 minute.

14. Take the liquid back to the spin column and centrifuge them for one more time.

15. Store the liquid which is the plasmid solution under -20℃.

Restriction enzyme digestion

1. Prepare the digestion system

Digestion System	20μL	50μL
Enzyme 1	1	1.5
Enzyme 2	1	1.5
10* Buffer	2	5
pUC-19-Amp	Less than 1μg	Less than 1μg
H₂O	Up to 20μL	Up to 50μL

2. Culture the system under 37℃ for 3-6h.

Electrophoresis

1. Add 10* Loading Buffer into sample at the rate of 1:10.

2. Add the sample into the slot of the gel.

3. Start electrophoresis. Run it for 40min to 1h.

Connecting

1. Prepare the connecting system

Connecting system	20	50μL
T4 DNA ligase	1μL	2.5μL
T4 Buffer	2μL	5μL
Target sequence	4 times to the volume of plasmid	Same as the left
plasmid	Less than 500ng	Less than 500ng
H₂O	Up to 20μL	Up to 50μL

Parts Examination

Enzymatic activity testing

1. Add 2-3mL blank medium into a clean cuvette.

2. Add 2-3mL bacteria liquid into another clean cuvette.

3. Take blank medium as the blank, examine the concentration of bacteria at 600nm in a VIS-Spectrophotometer.

4. Add 2-3mL bacteria liquid into a centrifuge tube, then centrifuge it at 12000r/min for 3 minutes.

5. Add 1mL upper liquid into a clean cuvette, then add 2mL more 0.1M HCl into it. Rotate the mixed liquid for several times to make sure it is well mixing.

6. Take blank medium as the blank, examine the concentration of riboflavin at 444nm in a VIS-Spectrophotometer.

Running Device

MFC assembly

1. Treatment of carbon fiber felt
The carbon fiber felt was first cleaned by deionized water，and was then boiled with 1mol/L NaOH and 1mol/L HCl each for 30 minutes. After each boiling,it was cleaned by deionized water until its pH value is 7. Finaly it was dried at 105。C for 20 minutes.

2. Treatment of proton exchange membrane
The proton exchange membrane was boiled in turn in 30% H2O2, deionized water, 0.5mol/L H2SO4 and deionized water for 30 minutes.

3. Sterilization
250ml graduated cylinders,2 250ml conical flasks,12 50ml centrifuge tubes,cathode chamber and anode chamber(with magnon),sterile water,gaskets and carbon fiber felt(fixed on titanium wire)

4. Combination of cathode chamber and anode chamber
A proton exchange membrane was put between the anode and the cathode,fixed with gaskets and parafilm.Check for leaks with sterile water.

5. Treatment of bacteria solution
Bacteria was cleaned with M9 medium, and the absorbance (OD values ) was measured in the 600nm wavelength and were adjusted to the 1.3--1.4.

6. Add liquids to cathode chamber and anode chamber

6.1anode chamber:

24ml bacteria solution after treatment,216ml M9 medium,appropriate amount 20mg/ml CuSO₄ mother solution (make sure the final concentration of Cu²⁺ are 0、1、2、3、4mg/L)

6.2cathode chamber:

240ml PBS（with 100mmol/L K₃[Fe(CN)₆]）

7. Fixed carbon fiber felt
Carbon fiber felt was put in front of the carbon fiber felt,and the titanium wire tied to it was fixed on the bottle cap of cathode chamber and anode chamber.

MFC operation

1. The MFC was put on the magnetic disk, the temperature and the speed of magnon were set, the temperature sensors were put into liquids.

2. Voltage was recorded every other minute.Find the relationship between peak voltage and the concentration of Cu²⁺.

FCM

Methods

1. We extracted the plasmid by the OMEGA^TM Plasmid Mini Prep Kit (D6943-02).

2. BBa_I13504 was cut by XbaI and PstI. J23101, J23106 and J23117 were cut by SpeI and PstI. The I13504 was inserted into the 3 vectors above by T4 Ligase. Finally the devices we constructed were transformed into E.coli DH5α.

Cultivation

3. The cultivation of our bacteria was performed in test tubes (16cm long, diameter is 3cm) filled with 5ml LB medium. The incubator was kept at 37°C and 260 rpm shaking frequency.

4. Chloramphenicol was added to each media. Chloramphenicol was added from a 1000X stock stored at -20°C for a final concentration of 25 µg/ml.

Measurement

5. Cells were washed by PBS twice in order to clean the medium.

6. Measurement was experimented via LSRFortessa, the channel was 488nm/510nm.

7. The data was exported by FCSDiva as .fcs files. If one cell's FITC value is higher than 103, we believe that the cell is colored. We calculated the ratio of colored cells in total cells. The ratio*100 was set as the unit of the device's strength.

Media

LB medium (1000mL)

1. Adding components

Tryptone	10g
Yeast extract	5g
NaCl	10g
H₂O	Up to 1000mL

2. Adjust the pH value to be around 7.2 by adding HCl or NaOH

3. Sterilize the medium under 121℃ for 20-25 minutes.

PS: adding agarose to the rate of 1.8%-2.0% before pH value adjustment to make it solid medium; adding antibiotic after sterilization when the medium is about 50-60℃ to make it selective medium.

M9 medium (1000mL)

1. Adding components

NH₄Cl	10g
Casein hydrolysate	5g
Na₂HPO₄ · 12H₂O	10g
KH₂PO₄	1g
H₂O	992ml

         2. Adjust the pH value to around 7.2 by adding HCl or NaOH.
         3. Sterilize the medium under 121℃ for 20-25 minutes.
         4. Add 10mL sterilized 20% glucose, and 1mL sterilized 1M MgSO4. The step must be operated in asepsis environment like clean bench

MM medium (1000mL)MM medium (1000mL)MM medium (1000mL)

1. Adding components

NH₄NO₃	1g
KH2PO4	0.5g
Na₂HPO₄ · 12H₂O	3.7g
NaCl	1g
MgSO₄·7h₂O	1g
H₂O	Up to 1000ml

2. Adjust the pH value to around 7.2 by adding HCl or NaOH.

3. Sterilize the medium under 121℃ for 20-25 minutes.

Kit&Buffer

OMEGA^TM Plasmid Mini Prep Kit (D6943-02)

OMEGA^TM Gel DNA Extraction Kit

TianGen^TM Gel DNA Extraction Kit

	EcoR 1	Xba 1	Spe 1	Pst 1
EcoR 1	1*H	1*M	1*H	1*H
Xba 1	1*M	1*M+BSA	1*M	1*M
Spe 1	1*H	1*M	1*M	1*H
Pst 1	1*H	1*M	1*H	1*H

All enzymes we used in the study was from TAKARA^TM.

Team:LZU-China/chz notes

Notes in Lab

A Novel Microbial Fuel Cell System that meaxure the content of estrogen

Journal

Parts Construction