Team:Hong Kong-CUHK/Laboratory log book

From 2011.igem.org

(Difference between revisions)

Latest revision as of 04:07, 3 October 2011

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Week 0

2 June2011 (Tue)

1. Frankie- transformed the bacteria with RFP andprepared the T7 and HR gene plasmid for PCR.

Week 1 Monday 6 June – Sunday 12 June

8 June2011 (Wed)

l test diffconc of NaCl to the survival of bacterial cell DH5a & BL21

1. Test the effect of different concentrationof NaCl to the survival of bacterial DH5a &BL21

9 June2011 (Thu)

l repeat +IPTG inducer
PCR amp. T7 and HR, gel clean to get T7 and HR gene from gel

1. Repeat Wed work

2. IPTG inducer

3. PCR amplification - T7 and HR(rhodopsin)

4. Gel clean to get T7 and HR gene from gel

10 June2011 (Fri)

1. Restriction cut of T7, HR and iGEM vector,ligate them together

2. Received all medium to grow magnetobacteria

Week 2 Monday 13 June – Sunday 19 June

13 June 2011 (Mon)

1. Nanodrop test DNAconcentration

2. Transformation tocompetent cells, spread on plate A & plate K

14 June 2011 (Tue)

1. no colonies were found on spread plate. possiblereasons:

O antibiotic C resistance spread on plate K

O failed restriction cut,

O spreading tools too hot which kill the bacteria…

2. Attempt to mix culturing medium for halobacterium salinarum DSM 3754,haloterrigena turkmenica DSM 5511, Natronomonas pharaohs DSM 2160, but couldn'tfind casamino acids which are essential for all the medium required

3. Repeat work oflast week: restriction cut, ligation

15 June 2011 (Wed)

1. transformation of E.coli competent cells.

Week 3 Monday 20 June – Sunday 26 June

20June (Mon)

1. Restriction cut

O pSB1A3 & pSB1K3

O HR & T7 promoter

O restriction enzyme: EcoR1 &Pst1

O incubate at 37^oC , 2hours

2. PCR purificationof cut product

O refer to kit protocol

3. Agarose gelelectrophoresis (Run gel) of purified product

O at 120V, 45minutes

O Failed. Possible reasons: insufficient DNA conc. Ingel, stock problem

4. Ligation

O HR+pSB1A3

O T7+ pSB1A3

O HR+ pSB1K3

O T7+ pSB1K3

O at 16^oC, overnight

5. Inoculationtransformation buffer preparation

O Steps of preparing 500ml inoculation transformationbuffer

1. 55mMMaCl₂‧4H₂O (5.4425g)

2. 15mMCaCl₂‧2H₂O (1.1025g)

3. 250mMKCl (9.3189g)

4. 10mMPIPES (10ml) (taken from 4^oC fridge)

5. Storedin 4^oC fridge

O 109 plate with antibiotic A and K is poured

6. Detection of T7and HR by gel electrophoresis

O no band of sample is shown

21 June (Tue) Norecord

22 June (Wed)

1. Pick colonies

O Each plate pick2 colonies into 2 tubes

2. Inoculation

O at 37^oC, 250rpm, 7hours

3. Transfer

O DH5α(B tube) :1ml & 0.5 ml

O BL 21 (B tube): 1ml & 0.5 ml

O Rosetta (Atube): 2ml

O Overnight,200rpm

4. Prepare 500ml LB, 500ml LB with 1M NaCl and500ml LB with 1M KCl solution LB solution

5. transformpET27bHR with K resistance into DH5α, BL21

23 June (Thu)

1. Measure OD oftransfer

O All tubes’ OD> 0.55

O Due tocontamination in the step of picking colonies. We shouldn’t put the pipette tipsinto the tubes since our medium does not contain antibodies

2. Repeated theexperiment from picking colonies and inoculation

O Only DH5α isused

O 7 hoursinoculation

O Inoculationfailed.

3. Restarted fromculturing E.coli on agar plate

O DH5α & BL21

4. Restriction cutand Double Digestion

O HR 20ng/ul

O T7 20ng/ul

O Since there arenot enough solution(a 50ul system should have at least 200ng of DNA) to make upa 50ul system, we modify the system to 25ul.

Solution (added by order)	Amount(ul)
ddH₂0	14
10X Buffer 3	2.5
10X BSA	2.5
EcoR1	0.5
Pst1	0.5
HR	5
Total	25ul

Solution (added by order)	Amount(ul)
ddH₂0	14
10X Buffer 3	2.5
10X BSA	2.5
EcoR1	0.5
Pst1	0.5
T7	5
Total	25ul

Solution (added by order)	Amount(ul)
ddH₂0	15
10X Buffer 3	2.5
10X BSA	2.5
EcoR1	0.5
Pst1	0.5
pSB1	4
Total	25ul

O The 3tubes areput at 37 ^oC for 2hours

5. Restriction cut

O T7/HRrestriction cut mixture

O pSB1 T3restriction cut mixture

O The mixture isput into thermomixer at 37oc for 2hs

O Inoculated 5mlstart culture of DH5α,, no BL21 colony is observed on the plate

O 6 sets ofsolution is prepared

0 – 1M NaCl with IPTG

0 – 1M NaCl without IPTG

0 – 1M KCl with IPTG

0 – 1M KCl without IPTG

0M salt withIPTG

0M saltwithout IPTG

(the solutionwithout salt is set as controls)

[Salt]/M	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	0.8	0.9	1.0
LB + salt/ml	0	0.4	0.8	1.2	1.6	2.0	2.4	2.8	3.2	3.6	4
LB/ml	4.0	3.6	3.2	2.8	2.4	2.0	1.6	1.2	0.8	0.4	0

O For each tube,2ul IPTG is added (for sample with IPTG), 40ul DH5α start culture, 4ml LB+ salt volumes

O 5ml start culture of DH5αisinoculated again for the miniprep on next day

O OD of solution is checked on nextday

24 June (Fri)

1. Picking E.colicolonies cultured yesterday

O DH5α & BL21

2. Inoculation:

O 4 DH5α tubes& 4 BL21 tubes

O Each with 5mlLB

O 37℃, 250rpm

3. Transferinoculation mixture into 125ml LB in conical flask

O 1.0ml DH5α-2,0.5ml DH5α-2

O 1.0ml BL21-2,0.5ml BL21-2

O Shake at 37℃, 250rpm, overnight

4. Transform“HR+1T3” & “T7+1T3” into DH5α competent cells

O 1.0ml HR+1T3,0.5ml HR+1T3

O 1.0ml T7+1T3,0.5ml T7+1T3

O 1 control platewithout antibiotics tetracycline

O Incubate at 37℃, overnight

5. Light absorbance of DH5α KCl IPTG

O DH5α KCl, DH5αNaCl IPTG and DH5α NaCl is measured

Light absorbance of DH5α KCl IPTG
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(5x)	0.532	2.660
0.2(5x)	0.515	2.575
0.3(5x)	0.477	2.385
0.4(5x)	0.390	1.950
0.5(5x)	0.370	1.850
0.6(5x)	0.267	1.335
0.7(5x)	0.92	0.960
0.8(2x)	0.300	0.600
0.9(2x)	0.156	0.312
1.0	0.249	0.249
0	0.791	0.791

Light absorbance of DH5α KCl
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(5x)	0.500	2.500
0.2(5x)	0.447	2.235
0.3(5x)	0.430	2.150
0.4(5x)	0.399	1.995
0.5(5x)	0.305	1.525
0.6(5x)	0.264	1.32
0.7(5x)	0.205	1.025
0.8(5x)	0.117	0.585
0.9(2x)	0.220	0.440
1.0	0.263	0.263
0(5x)	0.519	2.595

Light absorbance of DH5α NaCl IPTG
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(2x)	0.829	1.658
0.2(2x)	0.844	1.688
0.3(5x)	0.898	1.796
0.4(5x)	0.384	1.920
0.5(5x)	0.340	1.700
0.6(5x)	0.208	1.040
0.7(2x)	0.393	0.786
0.8(5x)	0.287	0.574
0.9(2x)	0.119	0.238
1.0	0.113	0.113

Light absorbance of DH5α NaCl
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(5x)	0.499	2.495
0.2(2x)	0.478	2.390
0.3(5x)	0.513	2.565
0.4(5x)	0.390	1.950
0.5(5x)	0.302	1.510
0.6(5x)	0.246	1.230
0.7(5x)	0.194	0.970
0.8(2x)	0.269	0.538
0.9	0.284	0.284
1.0	0.103	0.103

6. Miniprep

7. Storage of cells

O Transfer1ml from each cell culture to 1.5ml microcentrifuge tube

O Spinfor 1min

O Removemedium by discarding and pipetting

O Storethe cells at -80^oc

25 June (Sat)

1. OD measurement(OD 600nm) of transferred product

BL21	0.5ml	1.891
BL21	1.0ml	1.831
DH5α	0.5ml	0.670
DH5α	1.0ml	1.415

O All tubes’ OD> 0.55

O Might havecontamination

O Still used DH5α0.5ml 0.670 OD600 to make competent cells

O Made 25 tubesof competent cells at -80℃

2. pick colonies, Inoculation,

O 2 from HR+1T3,2 from T7+1T3 II, 1 from HR+1T3 I into 5ml LB+tetracycline snap-cap tube

3. Transfer 125mlmagnetotactic bacteria medium to a conical flask, waiting for autoclave

Week 4 Monday 27 June – Sunday 3 July

27 June (Mon)

1. Inoculated5ml start culture of DH5αtransformed with HR pET37b using the plate the week before

2. Prepared 6conical flasks

O LB with 50ul IPTG + 100ulantibiotic K

O LB without IPTG + 100ulantibiotic K

O 0.6M NaCl LB with 50ul IPTG +100ul antibiotic K

O 0.6M NaCl LB without IPTG + 100ulantibiotic K

O 0.6M KCl LB with 50ul IPTG +100ul antibiotic K

O 0.6M KCl LB without IPTG + 100ulantibiotic K

O Formeasuring OD absorbance and plot growth curve of DH5α on 28/06/2011 every 30/45min

3. Prepared 4 setsof solution same as 23/06/2011 to verify the optimum concentration of salt for DH5α to grow and absorb salt and added DH5α start culture

4. Inoculate 5mlstart culture of DH5α transformed with HR pET27busing the plate of the week before for the 6 conical flasks for 8/06/2011growth curve (+5ul antibiotic K)

28 June (Tue)

1. Prepare another set of competent cell

O Pick single colony from plates prepared onSat

O Transfer the colony into 5ml of LB broth inFALCON

O Incubate for 6hrs @37℃, shaking (250-300rpm)

O ~6pm, use the incubated starter culture toprepare 2 1L flasks

FLASK1: 0.5ml culture + 125LB

FLASK2: 0.25 ml culture + 125LB

O Incubate at 22℃,moderate shaking, overnight

2. Transform competent cells prepared on Sat 25June

O Incubate at 37℃, overnight

3. Plasmid DNA purification

O T7, PSB1K3 / HR, PSB1K3

O USING Spin MiniPrep Kit and aMicrocentrifuge

4. Lightabsorbance of DH5α KCl IPTG, DH5α KCl, DH5α NaCl IPTG, DH5α NaCl, DH5α 0M LB and DH5α 0M LB IPTG

Light absorbance of DH5α KCl IPTG
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(2x)	0.916	1.832
0.2(2x)	0.756	1.512
0.3(2x)	0.527	1.054
0.4	0.426	0.426
0.5	0.710	0.710
0.6	0.310	0.310
0.7	0.156	0.156
0.8	0.129	0.129
0.9	0.072	0.072
1.0	0.012	0.012

Light absorbance of DH5α KCl
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1	0.618	2.472
0.2	0.427	1.708
0.3	0.274	1.096
0.4	0.845	1.690
0.5	0.474	0.948
0.6	0.970	0.970
0.7	0.626	1.252
0.8	0.504	0.504
0.9	0.083	0.083
1.0	-0.011	-0.011

Light absorbance of DH5α NaCl IPTG
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(2x)	0.794	1.588
0.2(2x)	0.716	1.432
0.3(2x)	0.483	0.966
0.4(2x)	0.436	0.872
0.5	0.761	0.761
0.6	0.713	0.713
0.7	0.641	0.641
0.8	0.491	0.491
0.9	0.168	0.168
1.0	0.044	0.044

Light absorbance of DH5α NaCl
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(2x)	0.932	1.864
0.2(2x)	0.719	1.438
0.3(2x)	0.535	1.070
0.4(2x)	0.525	1.050
0.5	0.882	0.882
0.6	0.819	0.819
0.7	0.712	0.712
0.8	0.470	0.470
0.9	0.123	0.123
1.0	0.031	0.031

Light absorbance of DH5α 0M LB
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1	0.763	1.526
0.2	0.829	1.658

O 0.8mlDH5α is added to startculture solution of the 6 conical flask prepared the day before, the absorbanceof solution is measured per 30mins

Time(h)\OD of Solution	LB	LB IPTG	0.6M NaCl LB	0.6M NaCl LB IPTG	0.6M KCl LB	0.6M KCl LB IPTG
0	0.015	0.016	0.028	0.030	0.025	0.047
0.5	0.025	0.024	0.050	0.017	0.041	0.014
1	0.035	0.039	0.077	0.015	0.025	0.029
1.5	0.071	0.080	0.070	0.005	0.036	0.026
2	0.146	0.176	0.111	0.005	0.044	0.044
2.5	0.307	0.338	0.148	0.003	0.063	0.057
3	0.473	0.527	0.262	0.017	0.093	0.093
3.5	0.650	0.669	0.345	0.012	0.134	0.137
4	0.874	0.888	0.473	0.003	0.219	0.210
4.5	10.66	1.128	0.626	0.001	0.323	0.310
5	0.753(2x)	0.800(2x)	0.722	0.001	0.386	0.365

O 5mlstart culture of DH5α istransformed with HR pET27b (using the plate the week before) is inoculated, 6conical flasks of solution is prepared same as the 12/06/2011

29 June (Wed)

1. Measure OD of competent cell preparedyesterday

Flask	1	2	3	4
OD 600	1.697	1.844	1.840	1.662

O All flasks have OD greater than 0.55, itindicates contamination.

O Failed

2. Measure DNA concentration

O Use DNA purified yesterday

	260/280	DNA concentration (ng/ul)
T7 pSB1K3 (1)	1.95	32.6
T7 pSB1K3 (2)	1.90	32.2
HR pSB1K3 (1)	2.08	12.2
HR pSB1K3 (2)	2.01	12.3

3. Prepare new competent cells

O Transfer 0.1 nd 0.5ml DH5a and BL21 into125ml LB

4. Double restriction cut of T7 pSB1K3 and HRpSB1K3

Water	11.75ul
Buffer 3	3.5ul
BSA	0.35ul
EcoR1	0.7ul
Pst1	0.7ul
T7 pSB1K3	8ul
Total	25ul

Buffer 3	2.5ul
BSA	0.25ul
EcoR1	0.5ul
Pst1	0.5ul
HR pSB1K3	21.25ul
Total	25ul

O Incubate at 37oC for 2hrs

5. Run gel

O T7: band at ~150kb

O HR: band at ~800kb

6. The absorbanceof 6 solutions

Time(h)\OD of Solution	LB	LB IPTG	0.6M NaCl LB	0.6M NaCl LB IPTG	0.6M KCl LB	0.6M KCl LB IPTG
0	0.002	0.023	0.019	0.026	0.001	0.001
0.5	0.006	0.004	0.020	0.032	0.003	0.018
1	0.012	0.022	0.043	0.031	0.011	0.006
1.5	0.023	0.028	0.047	0.021	0.017	0.032
2	0.110	0.151	0.065	0.068	0.025	0.021
2.5	0.279	0.345	0.129	0.075	0.029	0.053
3	0.430	0.455	0.488	0.137	0.068	0.057
3.5	0.591.	0.640	0.230	0.183	0.089	0.112
4	0.748	0.830	0.310	0.264	0.122	0.150
4.5	0.962	1.053	0.445	0.353	0.183	0.265
5	0.747	0.832	0.389	0.310	0.177	0.209
5.5	0.939	10.17	0.448	0.380	0.235	0.261
6	0.764	0.745	0.360	0.316	0.251	0.310

30 June (Thur)

1. Measure OD of 0.1 and 0.05ml of DH5a andBL21 in 125 LB prepared on 29 June

	Volume (ul)	OD 600
DH5a	50	0.352
	100	0.523
BL 21	50	0.349
	100	1.710

O DH5a 100ul is taken for making competentcells

O Use competent cells made to transform T7 andHR

O Streak plates to check if competent cellsviable

O Total of 8 plates

O Incubate at 37^oC overnight

2. Spread plates with transformed competentcells

O T7 pSB1K3 in DH5a

O HR pSB1K3 in DH5a

O Total of 4 plates

3. PCR purification of iGEM plasmid

4. PCR purification of plasmid made

O 1K3, 1T3, 1C3

5. Run gel

O No result

1 July (Fri)

1. Check plates

O All 8 plates shown bacteria growth

O All 4 plates shown transformed cells growth

2. Pick colonies

O DH5a and competent cells with T71K3 andHR1K3

O Total of 12 snap caps

O Shake at 250rpm, 37^oC overnight

3. PCR amplification and purification of iGEMpsB1K3

4. Run gel

O No banding

2 July (Sat)

1. Mini prep

O DH5a HR x3

O DH5a T7 x3

O HR1K3 x3

O T71K3 x2

O One of the T71K3 didn’t form pallet after centrifugation

2. Digestion

Water	11.2ul
DNA	10ul
Buffer 3	2.5ul
EcoR1	0.5ul
Pst1	0.5ul
BSA	0.25ul
Total	25ul

O Incubate at 37^oC, 2hrs

O Remaining samples are stored at -20^oC

3. Run gel (with 2 setups)

Well	1	2	3	4	5	6
	ladder	T71K3 (1)	T71K3 (2)	HR(1)	HR(2)	HR(3)

O Result: Lane1 and 3 have bands of ~100-200bp

Land 4 to 6 has bands of 800bp

Well	1	2	3	4	5	6	7
	ladder	T7(1)	T7(2)	T7(3)	HR1K3(1)	HR1K3(2)	HR1K3(3)

O Result: Lane2 to 4 has bands of ~100-200bp

Lane 5to 7 has bands of ~800bp

3 July (Sun): No record

Week 5 Monday 4 July – Sunday 10 July

4 July (Mon)

1. Pick colonies

O BL21, 3 snap caps

O Incubate at 37oC, 250rpm

4. Prepare plates with antibiotics

O 25 plates with C

O 43 plates with K

5. Transfer incubated BL21 to 125ml LB, totalof 3 samples, each with 50ul BL21 added

O Incubate at37^oC overnight, 150rpm

6. 5mlstart culture of DH5α isinoculated and put into the incubator in common room

7. 6 conicalflasks is prepared

O LB IPTG

O 0.6M NaCl LB IPTG

O 0.6M KCl LB IPTG

O 0.1M – 1M NaCl IPTG

O 0.1M – 1M KCl IPTG

O LB without IPTG

5 July (Tue)

1. Prepare competent cells

	OD
BL21 (1)	1.958
BL21 (2)	1.918
BL21 (3)	1.907

O OD greater than 0.55, repeat the experiment

O Using BL21, we incubate cells from pickingcolony from plates

O Culture from tubes are transferred to theflask containing LB at 25^oC overnight, 130 rpm

2. PCR purification of T71K3 plasmid

O Using iGEM protocol

O Concentration of primers is 100pmol/ul,hence we have to dilute it to 30pmol/ul

3. Run gel using iGEM protocol and productprotocol

O After PCR amplification

Result: both show bandings beyond 100bp

iGEMprotocol shows banding at 2000kb

O After PCR purification

Result: iGEM protocol shows banding at 2000kb

4. Inoculation

5. 1ml DH5α start culture solution is addedto3 conical flasks prepared on 4/7.

O The absorbance and cell densityis measured every 30 minutes.

O Cell density at 2h and 2.5 h isfailed to be measured.

O Procedure of measuring cell density

i. 1ml solution is transferred to microcentrifuge tube, centrifuge for 1minand resuspend

ii. 10ul solution is transfer to both side of the hematocytometer and coverwith the cover slit

iii. observe the central square with 10x magnification under microscope andcount the number of cell

O Dilution is required (10fold, 100fold,1000fold .. ) for higher absorbance

O (1OD unit = 109 cell)

O Absorbance of 21 tubes is measured.

Light absorbance of DH5α NaCl IPTG( 4times diluted)
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1	0.701	2.804
0.2	0.786	3.144
0.3	0.680	2.720
0.4(2x)	0.947	3.788
0.5(2x)	0.603	2.412
0.6(2x)	0.538	2.152
0.7	0.334	1.336
0.8	0.083	0.332
0.9	0.025	0.100
1.0	0.022	0.088

Light absorbance of DH5α KCl IPTG( 4times diluted)
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1	0.893	3.572
0.2	0.906	3.624
0.3	0.780	3.120
0.4	0.445	1.780
0.5	0.621	2.484
0.6	0.083	0.332
0.7	0.218	0.872
0.8	0.040	0.160
0.9	0.010	0.040
1.0	0.027	0.108

O Light absorbance of 3 solutions

Time(h)\OD of Solution	LB IPTG	0.6M NaCl LB	0.6M KCl LB
0	0.025	0.070	0.054
0.5	0.043	0.065	0.044
1	0.117	0.068	0.067
1.5	0.185	0.041	0.059
2	0.389	0.069	0.064
2.5	0.634	0.085	0.081
3	1.039	0.107	0.111
3.5	1.586	0.188	0.181
4	0.334	0.274	0.268
4.5	0.364	0.350	0.328
5	2.412	0.432	0.450

O Celldensity (HR, IPTG, LBK, DH5α, pET27b HR)

Time(h)	Averaged cell density
0	1.5 x 10⁴
0.5	2.0 x 10⁴
1	1.3 x 10⁵
1.5	2.0 x 10⁶
2	-
2.5	-
3	8.95 x 10⁶
3.5	8.30 x 10⁹
4	1.145 x 10¹⁰
4.5	6.70 x 10⁹
5	6.10 x 10⁹

6 July (Wed)

1. Prepare BL21 competent cell

BL21	OD
1	1.094
2	1.263
3	1.263
4	1.163

O OD>0.55, preparation failed

2. Restriction cut of T71K3 prepared on 5July

Buffer 3	5ul
BSA	0.5ul
Eco R1	1ul
Pst 1	1ul
T71K3	10ul

3. Run gel to check the size of 2 sets of T71K3

O Both samples show banding of ~4000kb

4. Streak plate: BL21 X7

5. 5mlstart culture of DH5α isinoculated and put into the incubator in common room

6. 3 conicalflasks is prepared

O LB IPTG

O 0.4M NaCl LB IPTG

O 0.4M Kcl LB IPTG

7 July (Thu)
1. Pick colony of BL21 X4 andinoculation

O 37^oC, 250rpm

2. PCR amplification

O “Home-made” HR1K3, “Home-made” T71K3, stock1K3

O Using iGEM protocol

PCR supermix	45ul
Upper primer	1ul
Lower primer	1ul
Water	25ul
DNA	0.5ul
TOTAL	50ul

3. Transfer 50ul BL21 to 125ul LB

O Inoculated at 37^oC, 250rpm

4. Run gel

O Lane1: ladder

O Lane3: “Home-made” HR1K3

O Lane4: “Home-made” T71K3

O Lane5: stock 1K3

5. OD of BL21 in 125ml LB

	1	2	3	4
16:30	0.006	0.007	0.007	0.020
16:45	0.007	0.013	0.01	0.007
20:45	0.143	0.085	0.085	0.136
22:15	0.367		0.312

6. 5ml DH5α start culture solution is addedto3 conical flasks prepared on 5/7

O The absorbance and cell densityis measured every 30 minutes

7. 3 conicalflasks is prepared

O 100ml LB + 50ul IPTG + 100ulantibiotic K

O 60ml LB + 40ml KCl LB + 50ul IPTG+ 100ul antibiotic K

O 60ml LB + 40ml NaCl LB + 50ulIPTG + 100ul antibiotic K

Time(h)\OD₆₀₀ of Solution	LB IPTG	0.4M NaCl LB	0.4M KCl LB
0	0.033	0.031	0.025
0.5	0.037	0.032	0.017
1	0.078	0.013	0.033
1.5	0.122	0.056	0.047
2	0.310	0.102	0.096
2.5	0.423	0.157	0.156
3	0.620	0.255	0.256
3.5	0.893	0.398	0.427
4	1.186	0.590	0.621
4.5	1.632	0.859	0.803

O Celldensity of DH5α, pET27b HR, LB IPTG with K

Time(h)	Averaged cell density
0	3x10⁶
0.5	2.5 x10⁶
1	3.75 x10⁷
1.5	1 x10⁷
2	9.75 x10⁸
2.5	9.15 x10⁸
3	2.43 x10⁹
3.5	2.555 x10⁹
4	2.7875 x10⁹
4.5	2.225 x10⁹

8July (Fri)

1. Streak plate of Bl21 competent cell

O incubate at 37^oC

2. transformation of Pwt: BBa_E0044 (GreenFluorescent Protein deviated from jellyfish Aequeora Victoria wild-type GFPwith plasmid pSB1A3, in 2011 kit plate 1 well 14G)

O 10ul H20 added to well 14G

O DNA concentration was measured: 85.1ug/ul

O 1ul of DHA was added to DH5a competent cell

O 2 tubes of competent cell was used, onebeing taken from other bb (labeled as DH5a T) and one being prepared byoverselves (labeled as DH5a)

Week 6 Monday 11 July – Sunday 17 July

11 July (Mon)
1. iGEM plasmid stock check:

pSB1K3	~ 0ul
pSB1C3	< 1ul
pSB1T3	~4-5ul
pSB1A3	~4-5ul

O Insufficient quantity

O Bacterial amplification (plasmid+biobrick)of pSB1A3 (taken from iGEM kit plate 1, 1G)

O Use competent cell prepared on 30June

O Transformation: put pSB1A3 + biobrick intoDH5a

O Result refered to 13July

12 July(Tue)
1. Nano drop

T7 + 1K3	42.5 ng/ul
HR + 1K3	13.5 ng/ul

O pSB1T3 labeled to be 25ng/ul

2. Double digestion

O Incubated at 37^oC, 2hours

H2O	19.25ul
Buffer 2	2ul
BSA	2.5ul
Eco R1	0.5ul
Spe 1	0.5ul
T71K3	0.25ul
TOTAL	25ul

H2O	14.25ul
Buffer 2	2.5ul
BSA	0.25ul
Xbal 1	0.5ul
Pst 1	0.5ul
HR1K3	7ul
TOTAL	25ul

H2O	17.25ul
Buffer 2	2.5ul
BSA	0.25ul
Eco R1	0.5ul
Pst 1	0.5ul
pSB1T3	4ul
TOTAL	25ul

O Result: yield of product is too low

3. Prepare T plate

4. Bacterial amplification of backbone +biobrick (Continue experiment on 11July)

O red colonies observed, planned to do MidiPrep on 13July

5. 1ml DH5α start culture solution is pipetted to each conical flasks

O The LB IPTG solution is contaminated, OD andcell count is too high and out of the normal range.

O Frankie advised to use 500ml next time

O Brian will check if the cell count machineworks

O Preparation of NaCl, KCl DH5α should includeusing pipette to draw all medium away to avoid bacteria killed by ice.

Time(h)\OD₆₀₀ of Solution	LB IPTG	0.4M NaCl LB	0.4M KCl LB
0	0.180	0.049	0.025
0.5	0.234	0.021	0.020
1	0.301	0.041	0.024
1.5	0.377	0.047	0.055
2	0.606	0.127	0.114
2.5	0.744	0.209	0.212
3	0.916	0.324	0.331
3.5	1.125	0.472	0.449
4	1.289	0.673	0.725
4.5	1.432	0.881	0.886
5	1.533	10147	1.122
5.5	1.654	1.345	1.364

O Celldensity of DH5α, pET27b HR, LB IPTG with K

Time(h)	Averaged cell density
0	2.21x10⁷
0.5	8.3 x10⁸
1	7.9 x10⁷
1.5	3.6 x10⁸
2	3.035 x10⁹

13July (Wed)

1. Double digestion (repeat experiment ofyesterday)

O Modify the volume to minimize the amount ofinsert(T7, HR)

Buffer 2	2ul
BSA	0.2ul
Eco R1	0.5ul
Spe 1	0.5ul
T71K3	16.8ul
TOTAL	20ul

Buffer 2	2ul
BSA	0.2ul
Xbal 1	0.5ul
Pst 1	0.5ul
HR1K3	16.8ul
TOTAL	20ul

H2O	12.8ul
Buffer 2	2ul
BSA	0.2ul
Eco R1	0.5ul
Pst 1	0.5ul
pSB1T3	4ul
TOTAL	20ul

2. Run gel to check cut products

1	2	3	4	5	6	7	8
blank	HR+1K3	Ladder	T7+1K3	blank	pSB1T3	blank	blank

3. Transformation of HR+1K3, T7+1K3 prepared on28June

O plates spread with antibiotics K, incubateovernight

O plates labeled as “HR+1K3 prepared on 28June”and “T71K3 prepared on 28June”

4. Red colonies observed on plates prepared on11July

O Miniprep tomorrow

5. Gel check after double digestion and gelextraction

O Lane3: ladder

O Lane6: T7 (13.1 ng/ul) ~200kb

O Lane9: HR (25.5 ng/ul) ~800kb

O Lane12: 1T3 (2.3ng/ul)

O Gel result: weak banding

6. Ligation

T4 ligase buffer	2ul
T4 ligase	1ul
T7	8ul
HR	6ul
Vector (pSB1T3)	3ul
TOTAL	20ul

14July (Thu): no record

15July (Fri)

1. Transformation

O DH5a competent cell prepared on 30June

O 3 way ligation (T7- HR- 1T3)

2. Inoculation

O Red colonies on plates prepared on 17July

O 2 centrifuge tubes prepared, labeled as redcolony1 & 2

O Transferred 250ul to 125mL LB (1:500 ratio)

O Shake at 37^oC, 250rpm, overnight

O Labeled as red col 1&2

16July (Sat)

1. Mini prep

O DNA from red colony culture

O 4 tubes of DNA obtained

2. Digestion

DNA	10ul
10X Buffer 3	2.5ul
10X BSA	2.5ul
EcoR1	0.6ul
Pst1	0.6ul
H2O	8.8
Total	25ul

O Incubate at 37oC for 2hrs

3. Pick colonies of 3-way assemblyDH5a

O Incubate at 37^oC, 250rpm

17 July (Sun)

1. Run gel and gel recovery of red colony

2. Mini prep, run gel and gel recovery of 3-wayassembly

3. Run gel using digestion product prepared on16/7 (red colony)

4. Mini prep DNA of bacteria culture (3-wayDH5a)

O Total of 5 samples of 3-way 1T3 are made,each with 40ul

O Stored at -20^oC

O 10ul of each is used for digestion and rungel to check size

Water	11.25ul
DNA	10ul
Buffer 3	2.5ul
EcoR1	0.5ul
Pst1	0.5ul
BSA	0.25ul
Total	25ul

O Incubate at 37^oC for 2hrs

5. Gel extraction of gel with 1T3 backbone fromred conlony

O 4 tubes of backbone are made

O Store at -20^oC

6. Pick red colony of DH5a

O 4tubes of LB snap cap

O Incubate at 37^oC, 25rpm

7. Transfer to 125ml LB

O Shake at 37^oC overnight, 250rpm

8. Streak plate using red colony

O Plates with antibiotics A are used

O Store in incubator

Week 7 Monday 18 July – Sunday 24 July

18 July (Mon)

1. Nanodrop

3-way	260/280	ng/ul
1T3 (1)	1.88	56.3
1T3 (2)	1.91	71.5
1T3 (3)	1.91	33.5
1T3 (4)	1.96	20.8
1T3 (5)	1.87	30.1

1T3 backbone (1)	3.78	3.9
1T3 backbone (2)	1.91	5.8
1T3 backbone (3)	1.77	2.0
1T3 backbone (4)	1.42	1.9

2. Mini prep of DH5a with kit plate 1G (redcolony)

O 5 tubes are prepared labeled as 1G (1) to (5)

O Store at -20oC

O Nanodrop

	ng/ul
1G (1)	6.37
1G (2)	0.04
1G (3)	0.22
1G (4)	4.48
1G (5)	0.04

3. Run gel (3-way 1T3)

O Show banding at ~4000-5000

4. Gel recovery of 3-way 1T3

5. Double digestion of 3-way 1T3

6. Run gel

O No banding shown

7. Restriction cut of 1G (red colony)

8. Run gel 1G (red colony)

O Band at ~1000 and ~3000-4000

9. Transformation

Plasmid: T71K3 and HR1T3

DH5a competent cells prepared on 30/6

10. Spreadplates

11. Prepared 5 conical flasks with thefollowing components

O LB with 50μlIPTG + 100 μl Antibiotic K

O LB of 0.4 M NaCl with 50 μl IPTG + 100 μlAntibiotic K

O LB of 0.6 M NaCl with 50 μl IPTG + 100 μlAntibiotic K

O LB of 0.4 M KCl with 50 μl IPTG + 100 μlAntibiotic K

O LB of 0.6 M KCl with 50 μl IPTG + 100μl Antibiotic K

12. Transformed 5 ml DH5αwith pET27b start culture and put them into the incubator at 37°Cin the common room.

19 July (Tue)

1. Pick colonies that we spread yesterday

O T71K3 (with antibiotics K added) x2

O HR1T3 x2

2. Inoculation at 37oC, 250rpm for 6hrs

O Failed

3. Add 500 μl DH5αstart culture into 5 respective conical flasks prepared on 18/7

O recordthe OD600 of 5 conical flasks and the results are recorded.

Time (h)	LB+IPTG	0.4M NaCl +LB +IPTG	0.6M NaCl +LB +IPTG	0.4M KCl+ LB+ IPTG	0.6M KCl + LB +IPTG
0	0.008	0.013	0.013	0.011	0.004
0.5	0.023	0.021	0.021	0.027	0.017
1.0	0.025	0.013	0.015	0.009	0.005
1.5	0.049	0.026	0.010	0.012	0.005
2.0	0.083	0.017	0.011	0.013	0.003
2.5	0.209	0.033	0.015	0.045	0.010
3.0	0.382	0.107	0.058	0.107	0.054
3.5	0.567	0.234	0.082	0.177	0.062
4.0	0.783	0.329	0.126	0.288	0.128
4.5	0.973	0.485	0.165	0.432	0.206
5.0	1.214	0.673	0.258	0.662	0.252
5.5	1.454	0.906	0.386	0.845	0.336
6.0	1.554	1.132	0.450	1.078	0.457

4. Transformed 3 start cultures of DH5αwith pET27b with the total volume of 5ml with the following components

O 5ml LB + 5μl K

O 3ml LB + 2ml of 1M NaCl + 5 μl K (LB with 0.4M of NaCl)

O 2ml LB + 3ml of 1M NaCl + 5 μl K (LB with 0.6M of NaCl)

20 July (Wed)

1. Pick colony

O HR1T3 X1

O T71K3 X1

O Red colony X3

O All with antibiotics added

2. Incubation

O 37^oC, 250rpm, 6hours

3. Transfer

O 37^oC, 240rpm, overnight

4. Prepared the autoclaved the 4 solutions

O 500 ml LB X2

O 500 ml LB constituting 1M NaCl X1

O 500 ml LB constituting 1M KCl X1

5. Prepared 5 conical with the followingconstitute

O LB with 50μl IPTG

O LB of 0.4 M NaCl with 50 μl IPTG

O LB of 0.6 M NaCl with 50 μl IPTG

O LB of 0.4 M KCl with 50 μl IPTG

O LB of 0.6 M KCl with 50 μl IPTG

6. Transformed 5 DH5α with pET27b startsolution with different NaCl and KCl concentrations

O 5ml LB + 5 μl antibiotic K

O 5ml of 0.4M NaCl + 5 μl antibiotic K

O 5ml of 0.6M NaCl + 5 μl antibiotic K

O 5ml of 0.4M KCl + 5 μl antibiotic K

O 5ml of 0.6M KCl + 5 μl antibiotic K

21July (Thu)

1. Midi Prep of red colony (1G) & HR

O 3 samples of 1G: 35.7ng/ul, 27ng/ul,124ng/ul

O 1 sample of HR: 5.0ng/ul

2. Digestion of 1G red colony using 1G(1) &1G(3)

O 1G(1)

Buffer 3	2.5ul
100X BSA	0.25ul
Eco R1	0.5ul
Pst 1	0.5ul
DNA	14ul (500ng)
H20	7.25ul

O 1G(3)

Buffer 3	2.5ul
100X BSA	0.25ul
Eco R1	0.5ul
Pst 1	0.5ul
DNA	4ul (500ng)
H20	17.25ul

O Incubated at 37^oC, 2hours

3. Run gel using digestion products

O Band size: ~1000 and 2000-2500kb

4. Could not measure the absorbance of LB of0.6M NaCl, 0.4M KCl and 0.6M KCl because the start culture of DH5αcannot grow properly. The absorbance of start culture of LB and LB of 0.4M NaClprepared on 20/7/2011 without adding DH5α were measured.Other than that, we also measured the absorbance of LB+ IPTG and LB of 0.4MNaCl+ LB + IPTG

O Result

	LB + IPTG	LB of 0.4M of NaCl
Start culture absorbance (1ml)	1.323	0.464

5.

Time (h)	LB+ IPTG	LB of 0.4M NaCl + IPTG
0	0.021	0.014
0.5	0.026	0.024
1.0	0.045	0.033
1.5	0.043	0.036
2.0	0.133	0.064
2.5	0.245	0.105
3.0	0.359	0.159
3.5	0.555	0.228
4.0	0.720	0.303
4.5	0.927	0.466
5.0	0628 (2X)	0.618
5.5	0.846(2X)	0.815
6.0	0.899(2X)	0.610(2X)

22July (Fri)

1. Run gel (HR)

2. Transformation

O 3A pSB1C3

O 5A pSB1K3

O 7A pSB1T3

O T7 1K3

O DH5a competent cells

3. Spread plate

O 3C plates of 3A-pSB1C3

O 4K plates of 5A-pSB1K3

O 4T plates of 7A-pSB1T3

O 3K plates of T7-1K3

23July (Sat)

1. Digestion using 1G(3) prepared on 21July2011

O incubated at 37^oC, 2hours

O product stored in 4^oC fridgewith label 1G backbone 23h

2. Pick colony & Inoculation

O using plates prepared on 22July

- 3ApSB1C3

- 5ApSB1K3

- 7A pSB1T3

- T7-1K3

O 3 snap caps containing 5ml LB andcorresponding antibiotics are prepared for each type of plate.

- 3ApSB1C3 23/7 莊

- 5ApSB1K3 23/7 莊

- 7ApSB1T3 23/7 莊

- T7-1K323/7 莊

- Snapcaps are put in common shaker

24July (Sun)

1. Mini prep using cultures prepared yesterday,24 samples are made:

O Stored in -20^oC fridge

- 3A1C3(1-8)

- 7A1T3(1-4)

- 5A1K3(1-4)

- T71K3(1-8)

Week 8 Monday 25 July – Sunday 31 July

25 July (Mon)

1. Restriction cut

O 1G (1A3)

2. Run gel to extract backbone 1A3

O 2 bands observed: ~1000 and ~2000-3000

3. Gel recovery

4. Nanodrop of 24 samples made on 24/7

	ng/ul		ng/ul		ng/ul
3A1C3 (1)	58.7	5A1K3 (1)	48.0	T71K3 (1)	47.6
3A1C3 (2)	42.8	5A1K3 (2)	24.2	T71K3 (2)	46.8
3A1C3 (3)	51.8	5A1K3 (3)	12.1	T71K3 (3)	47.6
3A1C3 (4)	58.1	5A1K3 (4)	12.0	T71K3 (4)	33.9
3A1C3 (5)	29.0	7A1T3 (1)	35.2	T71K3 (5)	47.1
3A1C3 (6)	28.6	7A1T3 (2)	24.2	T71K3 (6)	43.6
3A1C3 (7)	13.9	7A1T3 (3)	27.6	T71K3 (7)	41.4
3A1C3 (8)	34.9	7A1T3 (4)	27.1	T71K3 (8)	31.9

5. Transformedcompetent DH5α cells with vector pET27b HR and then spread them onK plate. The plate was then put into the incubator at 37°C

26 July (Tue)

1. Restriction cut using HR and T7

Buffer 3	2.5ul
BSA	0.25ul
EcoR1	0.5ul
Pst1	0.5ul
DNA (35.3ng/ul)	14.25ul
H₂O	7.0ul
Total	25ul

Buffer 3	2.5ul
BSA	0.25ul
EcoR1	0.5ul
Pst1	0.5ul
DNA (180ng/ul)	21.25ul
Total	25ul

O Incubate at 37oC for 2hrs

2. Run gel

O Result: HR:~800bp

T7:200bp

3. Gel recovery

4. 3-way assembly

H2O	11ul
10X T4 DNA ligase buffer	2ul
T4 DNA ligase	1ul
HR	2ul
T7	2ul
1A3	2ul
Total	20ul

5. Prepared 5 conical flasks

O LB with 50μl IPTG

O LB of 0.4 M NaCl with 50 μl IPTG

O LB of 0.6 M NaCl with 50 μl IPTG

O LB of 0.4 M KCl with 50 μl IPTG

O LB of 0.6 M KCl with 50 μl IPTG

6. 5 start cultures were also prepared

O 5ml LB + 5 μl antibiotic K

O 5ml of 0.4M NaCl + 5 μl antibiotic K

O 5ml of 0.6M NaCl + 5 μl antibiotic K

O 5ml of 0.4M KCl + 5 μl antibiotic K

O 5ml of 0.6M KCl + 5 μl antibiotic K

27 July (Wed)

1. Transform terminator (iGEM plate2 24C) intoDH5a

2. Restriction cut

O HR1K3 and HR-T71A3 (3-way assembly) by EcoR1and Pst1

3. Run gel

O Ladder mix together, cannot see size

4. Spread plates

O 6 K-plates of 24C terminator

5. Measured the absorbance of the 5 conicalflasks prepared on the 26/7 by cell counting machine with the following result

Start culture

LB

KCl 0.4M

NaCl 0.4M

KCl 0.6M

NaCl 0.6M

First measurement (4X)

0.430

0.674

0.410

0.380

Second measurement

(4X)

0.401

0.636

0.431

0.488

0.348

O Preciously, we use the qViro cell counter

Result

Time (h)/ culture	LB	KCl 0.4M	NaCl 0.4M	KCl 0.6M	NaCl 0.6M
0	0.023	0.046	0.027	0.022	0.025
0.5	0.025	0.038	0.029	0.029	0.027
1.0	0.036	0.032	0.026	0.039	0.048
1.5	0.078	0.038	0.035	0.043	0.041
2.0	0.142	0.104	0.057	0.038	0.040
2.5	0.295	0.154	0.113	0.053	0.053
3.0	0.437	0.287	0.179	0.077	0.067
3.5	0.601	0.408	0.270	0.126	0.104
4.0	0.796	0.580	0.373	0.162	0.146

28 July (Thur)

1. Collect plates from 27/7, store at 4^oC

2. Run gel

O 3-way assemblt plasmid made yesterday

O No bands shown

3. Restriction cut of pSB1A3

Buffer 2	2.5ul
BSA	0.25ul
EcoR1	0.5ul
Pst1	0.5ul
H₂O	16ul
DNA	5.25ul
Total	25ul

O 37oC for 2hrs

4. Run gel

O No bands shown

5. Measured the absorbance change over time ofthe flask LB IPTG (100ml) with DH5α transformed with pET27b measuredand counted the cells in CSLB G12

Result

Time (h)	OD₆₀₀	Cell Count (particle per ml)
0	-0.004	4.5 X 10⁸
0.5	-0.002	5.2 X 10⁸
1.0	±0.000	6.7 X 10⁸
1.5	+0.002	8.7 X 10⁹
2.0	+0.007	1.8 X 10⁹
2.5	+0.026	1.8 X 10⁹
3.0	+0.041	3.1 X 10⁹
3.5	+0.061	4.8 X 10⁹

29July (Fri)

1. Run gel

O 45min,120V

O 6Xloading dye: 4ul, DNA: 20ul

Lane	1	2	3	4	5	6	7	8
	ladder		1G cut	T7 cut	HR cut

O Result
- lane 3: 1000-1500bp & 2000-3000bp
- lane 4: 100-200bp
- lane 5: 500-1000bp

2. Gel clean of T7, HR, 1G

O Labeled: “T7 28/7 Mo”, “HR 28/7 Mo”, “1G29/7 Mo”

O Stored in 4^oC fridge

3. Ligation using T7, HR, 1G

O Incubated at 16^oC

HR	7ul
T7	7ul
1G	2ul
Ligation buffer	2ul
T4 ligase	1ul
H2O	1ul
TOTAL	20ul

4. 100mM MQAE solution and alinquoted into1.5ml black microcentrifuge tube with 500μl each stored at-20°C.5μlMQAE and 95μl salt solution were added to microplate andmicroplate readers was used in SC 127.

O MQAE

Mass (mg)	100
Molar mass (g mol^-1 )	326.1893
Concentration (mM)	100
Volume made (ml)	3.066

O Concentration of the salt solution with2-fold dilution

i. 100mM NaCl

ii. 50mM NaCl

iii. 25mM NaCl

iv. 12.5mM NaCl

v. 6.25mM NaCl

vi. 3.125mM NaCl

O Final concentration of MQAE in microplatereader is 5mM

30July (Sat)

1. PCR amplification of T7 & HR

O Labeled as T7 1, T7 2, HR

31July (Sun)

1. Run gel using PCR product

2. Gel recovery

O 1 tube of HR, 1 tube of T7, each of 30ul

3. Digestion

O 1G, 3A1C3, 5A1K3, 7A1T3

O Use products to run gel, obtain backbone

4. Ligation

O (HR, T7) & (1C3, 1K3, 1T3 backbone)

O 4 samples: T71K3, T71T3, HR1K3, HR1C3

O Incubated at 16^oC, overnight

5. Transformation

O use competent cell prepared on 30/6

O 4 samples: 3A1C3, 3A1T3, 5A1K3, 1G

O Spread plates: using A,K,C,T plates, each type2 plates

Week 9 Monday 1 Aug – Sunday 7 Aug

1Aug (Mon)

1. PCR amplificationof T7 and HR

2. PCR purificationof T7 and HR

3. Transformation

O 1G, 3A, 5A, 7A

-Used DH5acompetent cells

-2sets are prepared

O HR1C3, HR1K3, T71T3, T71K3

-Used DH5acompetent cells

-2sets are prepared

O chloride sensor

-Used DH5acompetent cells

-1tube is prepared

4. Spread plate

O One plate is used for each tube of transformedcompetent cells

O All plates stored in incubator at 37^oC

5. Run gel to checksize

O Result: HR:~800

T7: ~100-200

6. Prepared 1 flask comprising 100ml LB, 50μlIPTG and 100μl antibiotic K and also 1 DH5αwith pET27b HR start culture

2Aug (Tue)

1. Inoculation

O HR1K3 (failed)

O HR1C3

O T71K3 (failed)

O T71T3

O 3A

O 5A

O 7A

O 1G

O Chloride sensor (failed)

2. Restriction cut

O HR, T7, 1T3

3. Ligation

O HR1T3and T71T3

O Storeat -16^oC overnight

4. Transfer to 125ml conical flask for MIDIPre

5. Transformation

O 24C terminator and DH5a used

O each spread on 2 plates

6. Prepare agar plate

O C plates x18

O A plates x20

O K plates x21

7. Absorbance of the start culture wasmeasured and the cells were counted

Time (h)	OD₆₀₀	Cell count (cell/ml)
0	0.000	4 X 10⁸
0.5	0.003	1.4 X 10¹⁰
1.0	0.008	8.6 X 10⁷
1.5	0.022	3.3 X 10⁸
2.0	0.142	3 X 10⁸
2.5	0.203	7 X 10⁸
3.0	0.263	/

Result

O Brian suggested that the abnormality of the cellcount data might be due to the growing size of the bacteria. At 3.0 hour,signal could not be obtained as well as the reason.

3Aug (Wed)

1. Mini prep

O 3A

O 5A

O 7A

O T71T3

O Cl sensor

2. 3-way assembly

	Ng/ul	260/280
3-way	12.8	1.93
Cl sensor	1.2	1.85
T71T3	13.0	1.93
HR1C3	103.0 (15.9)	1.33
5A	35.3	1.87
7A	24.2	1.90

3. Concentration of the NaCl solution wasprepared as follow by serial dilution

O 1000mM NaCl

O 500mM NaCl

O 100mM NaCl

O 50mM NaCl

O 25mM NaCl

O 12.5mM NaCl

O 6.25mM NaCl

O 3.125mM NaCl

4. Concentration of MQAE was also prepared asfollow by serial dilution

O 100mM MQAE

O 10mM MQAE

O 1mM MQAE

5. 95μl of NaCl solutions and 5 μlof MQAE solutions were added into the microplate in the position as shownbelow.

As a result, the final MQAE concentration

O A1-A4,B1-B4, C1-C4, D1-D4, E1-E4, F1-F4, H1-H4are 5mM

O A5-A8, B5-B8, C5-C8, D5-D8, E5-E8, F5-F8, H5-H8are 0.5mM

O A9-A12, B9-B12, C9-C12, D9-D12, E9-E12, F9-F12,H9-H12 are 0.05mM

O The plate with aluminum foil was wrapped and putin the incubator at 37°C for1 hour and measured emission by microplate reader

O Result

1	2	3	4	5	6	7	8	9	10	11	12
5480	5633	5428	5554	3013	3267	3058	3209	1249	1335	1341	1267
8990	9199	9483	9446	5235	5222	5241	4649	1545	1646	1634	1630
763	21493	21870	23048	11411	11549	11711	10767	2564	2513	2549	2413
25542	29517	27811	29312	14561	14986	14392	14646	3056	3018	2848	2839
30141	32470	33457	27985	17182	17219	14902	19335	3351	3318	2974	2931
37055	36110	39855	35193	18985	18717	18398	16975	3565	3349	3216	3366
34258	36477	37255	35236	18320	18588	18778	6182	3425	3211	3424	3265
36893	37123	37393	35904	13574	25312	19447	1334	3386	3529	3191	3364

A: 1000mM NaCl

B: 500mM NaCl

C: 100mM NaCl

D: 50mM NaCl

E: 25mM NaCl

F: 12.5mM NaCl

G: 6.25mM NaCl

H: 3.125mM NaCl

4Aug (Thu)

1. Miniprep

	ng/ul	260/280
24C (1)	44.1	1.77
24C (2)	59.7	1.76
T71K3 (1)	---	---
T71K3 (2)	41.7	1.87
HR1K3 (1)	18.5	1.92
HR1K3 (2)	29.5	1.78
3A (1)	---	---
3A (2)	50.7	1.88
5A (1)	44.4	1.85
5A (2)	---	---
1G (1)	142.9	1.86
1G (2)	52.4	1.89
7A	43.5	1.86

O 3A (1) is red while 3A (2) is milky

O 1G (1) is red while 1G (2) is milky

2. Double digestion

O T71K3

Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Spe1	0.5ul
T71K3 (prepared on 4/8)	16.8ul
Total	20ul

O HR1C3

Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Pst 1	0.5ul
HR1C3 (prepared on 3/8)	16.8ul
Total	20ul

O 7A

Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Pst 1	0.5ul
7A (prepared on 3/8)	4ul
H2O	12.8ul
Total	20ul

-37^oC,2hours

3. 3-way ligation

T4 ligase buffer	2ul
T4 ligase	1ul
T7	10ul
HR	4ul
Vector (7A)	3ul
Total	20ul

4. Inoculation

O Pick colony of white from T71T3, HR1T3

5Aug (Fri)

1. Nanodrop

Samples prepared on 4/8	ng/ul	260/280
HR1T3 (1)	10.3	1.98
HR1T3 (2)	12.2	1.91
T71T3 (1)	10.5	1.82
T71T3 (2)	12.4	1.86

2. Transformation

O used DH5a competent cells

O only 1 set is prepared using 3-way ligationproducts

O Platewas spreaded after transformation using T plates

(plateslabeled: 5/8transformation, 3A ligation)

O store st 37oC

3. Pick colonies

O pick from T71T3

O 3 colonies are picked

O inoculate for 12-16hrs

O 3 snapcap tubes, 37^oC, 250rpm

7Aug (Sun)

1. inoculationform plates HR-T7-1T3 prepared on 6/8

2. pick3 colonies to snap-cap tubes with 6ml LB, then shake at 250rpm, 37^oCfor 12-16 hrs

3. Preparedand inoculated four tubes of the start culture with the volume of 10ml , whichare 0.4M NaCl LB with light, 0.4M NaCl LB without light , LB with light, and LBwithout light. But we didn’t add IPTG.

4. MeasuredOD 600 of four start culture

	0.4M NaCl LB with light	0.4M NaCl LB without light	LB with light	LB without light
OD600 (2X)	0.230	0.401	0.388	0.690

5. Spinfour tubes of start culture separately to remove the LB/NaCl LB then store in-80°Crefrigerator.

6. Preparedfour tubes of start culture with the total volume of 10 ml, which 0.4M NaCl LBwith light, 0.4M NaCl LB without light, LB with light and LB without light andinoculated them overnight 5μl of 1M IPTG was added

Week 10 Monday 8 Aug – Sunday 14 Aug

8Aug (Mon)

1. Mini prep 3-way ligation products preparedon 7/8

Result

	ng/ul	260/280
3-way ligation	11.5	1.83
HR-T7-1T3 (1)	13.9	1.86
HR-T7-1T3 (2)	13.2	1.91
T71T3 (1), prepared on 6/8	29.4	1.52
T71T3 (2) , prepared on 6/8	7.3	1.69
T71T3 (3) , prepared on 6/8	5.4	1.74

O DNA ladder used is from Fermentas GeneRuler 1kbDNA, #SM1331

2. Run gel

3. Double digestion

Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Spe 1	0.5ul
T71T3 (prepared on 6/8)	16.8ul
Total	20ul

Buffer 2	2ul
BSA	0.2ul
XBa 1	0.5ul
Pst 1	0.5ul
HR1K3 (prepared on 4/8)	16.8ul
Total	20ul
Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Pst 1	0.5ul
3A (prepared on 4/8)	2ul
H2O	14.8ul
Total	20ul

O 37^oC, 2hrs

4. Ligation

T4 ligase buffer	2ul
T4 ligase	1ul
T7	7ul
HR	7ul
Vector pSB1C3 from 3-way	3ul
Total	20ul

O 16^oC overnight

5. MeasuredOD₆₀₀of the four start cultures of 10ml of IPTG made on 7/8 then spinfour tubes of start culture separately to remove the LB/NaCl LB

O Put in the -80°C refrigerator for storage.

6. Preparedagain four tubes of 10ml of start culture

O LB of 0.4M NaCl with light

O LB of 0.4M NaCl without light

O LB with light

O LB without light

O Inoculated them with the addition of 5μl of 1MIPTG.

7. MeasuredOD₆₀₀ of the four start cultures.

Result:

OD₆₀₀of the four start cultures of 10ml of IPTGmade on 7-8-2011

	LB of 0.4M NaCl with light	LB of 0.4M NaCl without light	LB with light	LB without light
OD₆₀₀(2X)	0.328	0.503	0.875	0.934

OD₆₀₀of the four start cultures were measured.

LB of 0.4M NaCl with light

LB of 0.4M NaCl without light

LB with light

LB without light

OD₆₀₀ (2X)

0.357

0.526

0.390

0.627

O Spin four tubes to remove the LB/LB NaCl

O Store in -80°C refrigerator.

9Aug (Tue)

1. Transformation of 3-way ligation cloneprepared on 8/8

2. Transformation of T71T3 mini prep productprepared on 6/8

3. Transformation of 3-way ligation preparedon 8/8

O All experiment were done following the standardtransformation protocol, except that for: 1. 2ul added, 2. 2ul added, 3. 5uladded

4. Spread plates with antibiotics C, Trespectively

5. Pick colony from T71T3, HR1T3, 3-wayligation plates from refrigerator and then inoculate overnight

6. We unfortunately make a mistake thatbecause the OD was measured and spinned all of the solution, whereas the startsolution should be spinned with accordance with the volume calculated from themeasurement of the OD. Therefore, the whole experiment needs to be redone.

10Aug (Wed)

1. Mini prep of T71T3, HR1T3, 3-way ligationfrom 9/8

	ng/ul	260/280
T71T3 (1)	111.0	1.84
T71T3 (2)	109.9	1.85
T71T3 (3)	147.9	1.90
HR1T3 (1)	103.0	1.89
HR1T3 (2)	98.9	1.85
HR1T3 (3)	107.6	1.84
3A (1)	101.2	.182
3A (2)	123.6	1.88
3A (3)	210.0	1.87

O all are using 50ul H2O to elute al the finalstep, except that all tubes of 3 had been added 30ul to increase concentration

2. inoculation

Pick3 colonies from 3 plates respectively, totally of 9, from

O 3-way ligation(8/8) after mini prep done on 9/8

O 3-way ligation(8/8) done on 9/8

O T71T3 (6/8) done on 9/8

Labeled as follows:

3-way post miniprep (1)

3-way post miniprep (2)

3-way post miniprep (3)

3-way (1)

3-way (2)

3-way (3)

T71T3 (1)

T71T3 (2)

T71T3 (3)

3. Inoculated 4 DH5α with pET27b HRstart cultures namely,

O LB + IPTG +K with light

O LB + IPTG +K without light wrapped with aluminumfoil

O LB + 0.4M NaCl + IPTG +K with light

O LB + 0.4MNaCl + IPTG +K without light wrapped with aluminum foil

11 Aug (Thu)

1. Miniprep of 9 tubes prepared on 10/8

Tube	Concentration (ug/ul)	260/280
3A Post mini prep (1)	96.1	1.91
3A Post mini prep (2)	41.6	1.96
3A Post mini prep (3)	100.5	1.85
3A (1)	104.7	1.85
3A (2)	68.8	1.87
3A (3)	74.7	1.96
T71T3 (1)	87.3	1.79
T71T3 (2)	115.8	1.85
T71T3 (3)	135.9	1.91

O all no. 3 tubes are eluted with 30ul 60^oC water to increase yield andconcentration

2. Transform plate 2 24C terminator

O use competent cell made on 30/6

O 24C terminator mini-prep on 4/8 by Eric

O Plate stored at 37^oC incubator, overnight

O Remaining transformed 24C terminator storedat -20^oC,
labeled as “ transf. 24C, 11/8,ii)

3. Sequencing

O 9 tubes prepared on 10/8, 9 tubes preparedon 11/8

4. Measured the OD₆₀₀ of the 4start cultures prepared on 10/8

	LB of 0.4M NaCl with light	LB of 0.4M NaCl without light	LB with light	LB without light
OD₆₀₀ (2X)	0.266	0.267	0.302	0.286

5. 10% SDS was diluted into 2% SDS by addingthe autoclaved 40ml of distilled water and 10ml of 10% SDS based on theinsensitivity of SDS

6. Measure the OD600 of the second set withoutIPTG

	LB of 0.4M NaCl with light	LB of 0.4M NaCl without light	LB with light	LB without light
OD₆₀₀ (2X)	0.094	0.096	0.350	0.355
Volume to be spinned (ml)	*All	*All	4.5	4.5

All volume to bespinned because the OD of NaCl sets are too small so the total volumes arestill inadequate. As a result, a back up set without IPTG by inoculation wasprepared and collected on 12-8-2011. It was found that the DH5α plate has beenused up for cline picking so later transformation and plate spreading is need.Besides, it was found that there is only little amount of 1000K anti-biotictoo.

12 Aug (Fri)

1. Inoculation

O pick 3 colonies from plate “24C terminatortransformed 11Aug”

2. Measured the OD600 of the back up set ofbacteria.

	LB of 0.4M NaCl with light	LB of 0.4M NaCl without light	LB with light	LB without light
OD₆₀₀ (2X)	0.459	0.290	0.768	0.847
Volume to be spinned (ml)	3.5	5.5	2	1.9

3. Pellets after spinning down were stored in-80°C refrigerator

O 2% of 100μl of SDS was used to lyse thecells in 99°C for 5 minutes

O Serial dilution of the bacteria in 2X, 4X,8X and 16X and of salt like previously done on 3/8 were done

O The MQAE and the solutions were added intothe microplate and incubated for an hour

4. Measured the MQAE fluorescence withmicroplate reader

O The data were abnormal and Frankie deducedthat it may be due to the bubbles of 2% SDS

13 Aug (Sat)

1. Miniprepof 24C terminator prepared on 12/8

O 3 tubesare labeled as “24C (1) 13/8”, “24C (2) 13/8”, “24C (3) 13/8”

Week 11 Monday 15 Aug – Sunday 21 Aug

15 Aug (Mon)

1. Transformed pET27bHR into DH5αand spread on a K plate

16 Aug (Tue)

1. InoculatedLB IPTF, 0.4M NaCl IPTG, 0.4M NaCl IPTG with light, 0.4M KCL IPTG and 0.4M KClIPTG without light with DH5α pET27b

17 Aug (Wed)

1. Measuredthe OD 600 of the back up set of the bacteria

set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀ (2X)	0.857	0.724	0.733	0.316	0.660
Volume to be spinned down (ml)	1.762	2.086	2.060	4.778	2.288

2. 2% ofSDS was spinned to lyse the cells in 99°C for 5 minutes with 100μleach, Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt likepreviously done was carried out. The MQAE and solutions were added into themicroplate and inoculated for an hour.

3. Measuredthe MQAE fluorescence with microplate reader

O The datawas also abnormal and Frankie deduced that it may be also due to the bubbles ofthe 2% SDS.

4. Inoculatedthe following tubes with DH5α pET27b

O LB +IPTG

O 0.4MNaCl + IPTG without light

O 0.4MNaCl + IPTG with light

O 0.4MKCl + IPTG without light

O 0.4MKCL + IPTG with light

O ( 10μl K and 5μ of IPTG)

18 Aug (Thu)

1. Rungel with PCR products (T7, HR)

O Result:HR did not show band with 800 bp

2. Rungel using PCR product (T7)

O Result:the samples show unclear result

3. PCRwith T7 & HR

4. PCRpurification of PCR product of step 3

5. Measuredthe OD600 of the back up set of the bacteria

set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.862	0.242	0.751	0.797	0.252
Volume to be spinned down (ml)	2.8	10	3.2	3.0	9.6

6. 2%of SDS was spin to lyses the cells in 99°C for 5 minutes with 100μl each,Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt likepreviously done was carried out. The MQAE and solutions were added into themicroplate and inoculated for an hour.

7. MQAEfluorescence was measured with microplate reader

O Thedata was abnormal and may be due to the bubbles of the 2% SDS.

19Aug (Fri)

1. Rungel to check size of PCR amplified T7 & HR prepared on 18/08

7. HR:result failed, at ~100bp

8. T7:result ok, at 100-200bp

20Aug (Sat)

1. Inoculationof 3 plate prepared last night

O 6 tubes prepared

O Abandoned because MidiPrep cannot be done onSunday, will do miniprep instead)

2. PCRwith HR templates (2tubes)

3. Restrictionvut T7 prepared on 18/8 & backbone 5A1C3

O 1tube of T7

Buffer	2ul
BSA	0.2ul
EcoR1	0.4ul
Pst1	0.4ul
T7	15ul
H20	2ul
TOTAL	20ul

O 2tubes of backbone

Buffer	2ul
BSA	0.2ul
EcoR1	0.4ul
Pst1	0.4ul
backbone	10ul
H20	7ul
TOTAL	20ul

4. PCRpurification of PCR product HR (2tubes) (continued step 2)

5. Rungel to check size of HR purified product

O Run 50ul of each HR and do gel recovery

O Each lane of HR with 25ul

O Result: size correct, ~800bp

Lane	1	2	3	4	5	6
	---	100bp ladder	HR-1	HR-1	HR-2	HR-2

6. Ligationof T7 & 1C3, overnight

O 3 tubes

T4 ligase buffer	2ul
T4 ligase	1ul
T7	3ul
1C3	14ul
TOTAL	20ul

7. Inoculationof 24C terminator DH5a

21 Aug (Sun)

1. Miniprep6 tubes of 24C terminator

2. Transformation:

O T71C3 (1) prepared on 20/8

O 1G1A3

O 3A1C3

O 5A1K3

O 7A1T3

O Spread plate:
- white colonies: T71C3
- red colonies: 1G1A3, 3A1C3, 5A1K3, 7A1T3

3. Restrictioncut of HR and backbone 1C3

O HR: 10ul

O Backbone X2: total 40ul

4. Ligationof HR & 1C3, overnight

O 3 tubes labeled as “HR1C3(1-3)”

5. Inoculatedwith DH5α pET27b

O LB + IPTG

O 0.4M NaCl + IPTG without light

O 0.4M NaCl + IPTG with light

O 0.4M KCl + IPTG without light

O 0.4M KCL + IPTG with light

O ( 10μl K and 5μ of IPTG)

Week 12 Monday 22 Aug – Sunday 28 Aug

22 Aug (Mon)

1. Inoculation

O 37^oC, 240rpm

O 1G1A3, 7A1T3 awhite colonies

O 3A1C3, 5A1K3ared colonies

O Did not pick T71C3

2. Nanodropof 24C (X6) & HR (X3)

	ug/ul	260/280
24C (1)	10.5	1.72
24C (2)	7.9	1.78
24C (3)	8.2	1.73
24C (4)	7.9	1.59
24C (5)	11.7	1.54
24C (6)	26.7	1.37
HR (1)	6	1.67
HR (2)	4.9	1.80
HR (3)	8	1.07

3. Transformation

4. Transferof 1G1A3, 7A1T3, 3A1C3, 5A1K3

O each type 2 tubes

5. inoculationof transferred product

6. MeasureOD600 of the back up set of the bacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.303	0.350	0.637	0.689	0.269
Volume to be spinned down (ml)	8	6.9	3.8	3.5	9

7. 2%of SDS was spin to lyses the cells in 99°C for 5 minutes with 400μl each

O Serial dilution of the bacteria in 8X, 16X,32X, 64X and of salt like previously done was carried out

O 95μl of each concentration was added intoeach well

O 5μl of MQAE was added into each wellafterwards

O A duplicate was made. Sample prepared byFrankie and 5 μl of MQAE were added with the same volume

8. Measuredthe MQAE fluorescence with microplate reader

O Resultof 0.4M NaCl IPTG without light lay in the range of our standards

9. Inoculatedthe following tubes with DH5α pET27b

O LB +IPTG

O 0.4MNaCl + IPTG without light

O 0.4MNaCl + IPTG with light

O 0.4MKCl + IPTG without light

O 0.4MKCL + IPTG with light

O ( 10μl K and 5μ of IPTG)

23 Aug (Tue)

1. midiprep3A1C3, 5A1K3

2. transform24C, chloride sensor, 1G1A3, 7A1T3,T71C3 with DH5a cells

O spread the above to agar plates

O each 2 plates

3. pickcolonies and inoculation of the above

O result: 1G1A3 & chloride sensor are toodense, others are ok

4. Measuredthe OD600 of the bacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.923	0.875	0.318	0.396	0.706
Volume to be spinned down (ml)	2.62	2.77	7.6	6.11	3.43

5. 2%of SDS was spinned to lyse the cells in 99°C for 5 minutes with 400μl each

O Serial dilution of the bacteria in 8X, 16X,32X, 64X and of salt like previously done was carried out

O 95μl of each concentration was added intoeach well

O A duplicate was made. Sample prepared byFrankie and 5 μl of MQAE were added with the same volume.

O MQAE fluorescence was measured by microplatereader and the results were sent to Frankie.

6. Routinely,the following tubes are inoculated with DH5α pET27b

O LB + IPTG

O 0.4M NaCl + IPTG without light

O 0.4M NaCl + IPTG with light

O 0.4M KCl + IPTG without light

O 0.4M KCL + IPTG with light

O ( 10μl K and 5μ of IPTG)

24 Aug (Wed)

1. miniprepHR1C3

O Result:

sample	Conc. (ng/ul)	260/280
HR1C3 (1)	30.5	1.74
(2)	24.7	1.81
(3)	19.9	1.94
(4)	22.1	1.97
(5)	28.3	1.80
(6)	50.0, 34.8, 21.0, 24.8, 25.3	1.94, 1.77, 2.25, 1.97, 1.91
(7)	42.1, 29.5	1.91, 1.92
(8)	24.6	1.78
(9)	31.4, 25.8	1.80, 1.91
(10)	18.9	1.91
(11)	19.2	1.99
(12)	48.2, 43.1, 45.8	1.88, 1.99, 1.92

2. transform1ul chloride sensor to 20ul DH5a competent cells

3. Measuredthe OD600 of the bacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.667	0.148	0.107	0.178	0.181
Volume to be spinned down (ml)	1.81	9.5	9.5	9.5	9.5

4. Thenwe use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μleach

O Serial dilution of the bacteria of 2-fold,4-fold, 8-fold and 16-fold was carried out

O The sample (A-I) prepared by Frankie wasadded into the well

O 95μl each and a duplicate was done

O Finally, 5μl of MQAE was added into thewells with the samples

O The microplate was put into the incubator at37°C for 1 hour.

5. Measuredthe MQAE fluorescence by microplate reader.

6. Followingtubes are inoculated with DH5α pET27b

O LB + IPTG

O 0.4M NaCl + IPTG without light

O 0.4M NaCl + IPTG with light

O 0.4M KCl + IPTG without light

O 0.4M KCL + IPTG with light

O ( 10μl K and 12.5μ of 0.4M of IPTG)

25 Aug (Thu)

1. Measured the OD₆₀₀of the bacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.851	0.718	0.767	0.322	0.717
Volume to be spinned down (ml)	3.4	4.0	3.8	9.0	4.0

2. Use 2% of SDS to spin to lyse the cells in99°C for 5 minutes with 400μl each

O Serial dilution of the bacteria of 2-fold,4-fold, 8-fold and 16-fold was carried out

O The sample (A-I) prepared by Frankie wasadded into the wells

O 95μl each and a duplicate was done

O Finally, 5μl of MQAE was added into thewells with the samples

O The microplate was put into the incubator at37°C for 1 hour.

3. Measured the MQAE fluorescence by microplatereader.

4. Following tubes are inoculated with DH5αpET27b

O LB + IPTG

O 0.4M NaCl + IPTG without light

O 0.4M NaCl + IPTG with light

O 0.4M KCl + IPTG without light

O 0.4M KCL + IPTG with light

O ( 10μl K and 12.5μ of 0.4M of IPTG)

26 Aug (Fri)

1. Measured the OD₆₀₀ of thebacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.878	0.326	0.447	0.262	0.625
Volume to be spinned down (ml)	2.7	7.2	5.3	9.0	3.8

2. Use 2% of SDS to spin to lyse the cells in99°C for 5 minutes with 400μl each

O Serial dilution of the bacteria of 2-fold,4-fold, 8-fold and 16-fold was carried out

O The sample (A-I) prepared by Frankie wasadded into the wells. 95μl each and a duplicate was done

O Finally, 5μl of MQAE was added into thewells with the samples

O The microplate was put into the incubator at37°C for 1 hour.

3. Measured the MQAE fluorescence by microplatereader.

4. Following tubes are inoculated with DH5αpET27b

O LB + IPTG

O 0.4M NaCl + IPTG without light

O 0.4M NaCl + IPTG with light

O 0.4M KCl + IPTG without light

O 0.4M KCL + IPTG with light

O ( 10μl K and 12.5μ of 0.4M of IPTG)

28 Aug (Sun)

1. restrictioncut of T7, HR (provided by Frankie) and plasmid 1C3

O HR+T7:
- HR cut by Xbal1 & Pst1
- T7 cut by EcoR1 & Spe1

O HR+1C3, T7+1C3: HR, T7, 1C3 cut by EcoR1& Pst1

2. ligationof HR & T7, HR & 1C3 backbone, T7 and 1C3 backbone respectively

O 10X T4 ligase buffer (2ul) , T4 ligase (1ul)for all tubes

O HR+T7: HR (8.5ul), T7(8.5ul)
- 2 sets, 1 set at 16^oC overnight, another set at room temperature, 1hour

O HR+1C3
- 2 sets, 1 set at 16^oC overnight, another set at room temperature, 1hour

O T7+1C3
- 1set, at 16^oCovernight

O Aliquot 3 tubes with 30ul 10X T4 ligationbuffer

O Use 10X T4 ligation buffer in 1.5mlmicrocentrifuge tubes

Week 13 Monday 29 Aug – Sunday 4 Sept

29Aug (Mon)

1. PCRpurification of ligation product

2. Rungel

Lane

1

2

3

4

5

6

7

Sample

1bb ladder

100bp ladder

HR T7

@16^oC

HR T7 @22^oC

T7 1C3 @16^oC

HR 1C3 @22^oC

HR 1C3 @16^oC

3. Nanodrop

4. Transformation

O Use DH5a and BL21 as competent cells foreach set

O T71C3 @16^oC

O HR1Ce@16^oC

O HR1C3@22^oC

O 6 spread plate stored at incubator

5. Followingtubes are inoculated with DH5α pET27b

O LB + IPTG

O 0.4M NaCl + IPTG without light

O 0.4M NaCl + IPTG with light

O 0.4M KCl + IPTG without light

O 0.4M KCL + IPTG with light

O ( 10μl K and 12.5μ of 0.4M of IPTG)

30Aug (Tue)

1. PCRpurification of HR1C3(16^oC), T71C3(16^oC),T7HR(16^oC)

2. Restrictioncut wth

O T7-HR: Spe1

O T7-HR: EcoR1 & Pst1

O 24C double terminator: Xbal1 & Pst1

O pSB1C3: EcoR1 & Pst1

3. PCRamplify and purify: HR, T7, HR-T7

4. Rungel to check size

O Result:
-HR, T7 a correctbands
-HR-T7 a no band

5. Nanodropof step 4 product

Sample	ng/ul	260/280
HR	12.4	1.70
T7	7.9	1.99
HR-T7	8.0	1.57

6. Ligationof step 2 restriction cut product

O (T7-HR) + (24C double terminator)

O (T7-HR) + (pSB1C3)

7. Spreadplate

O HR1C3 DH5a 16^oC

O HR1C3 BL21 22^oC

O T71C3 DH5a 16^oC

O T71C3 BL21 16^oC

O Chloride sensor

8. Measuredthe OD600 of the bacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.946	0.264	0.302	0.359	0.631
Volume to be spinned down (ml)	2.5	9.0	7.9	6.6	3.8

9. Use2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each

O Serial dilution of the bacteria of 2-fold,4-fold, 8-fold and 16-fold was carried out

O The sample (A-I) prepared by Frankie wasadded into the wells

O 95μl each and a duplicate was done

O Finally, 5μl of MQAE was added into thewells with the samples

O The microplate was put into the incubator at37°C for 1 hour.

10. Measuredthe MQAE fluorescence by microplate reader.

11. Followingtubes are inoculated with DH5α pET27b

O LB + IPTG

O 0.4M NaCl + IPTG without light

O 0.4M NaCl + IPTG with light

O 0.4M KCl + IPTG without light

O 0.4M KCL + IPTG with light

O ( 10μl K and 12.5μ of 0.4M of IPTG)

31Aug (Wed)

1. PCRpurification of ligation product

O (T7-HR)

O (T7-HR) + (24C double terminator)

O (T7-HR) + (pSB1C3)

2. Rungel

O (T7-HR) , (T7-HR) + (24C double terminator)ano band

O (T7-HR) + (pSB1C3)awrong size

3. Restrictioncut and ligation

O (T7-HR), HR1C3, T71C3

4. Rungel with ligation sample

O HR1C3acorrect size

O T7-HRano band

5. Gelrecovery with HR1C3

6. Pickcolony from plate prepared last night

O T71C3: DH5a

O HR1C3: DH5a & BL21

7. PCRamplification of HR & T7

8. Measuredthe OD600 of the bacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.301	0.588	0.454	0.269	0.468
Volume to be spinned down (ml)	8	4.1	5.3	9	5.2

9. Use2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each

O Serial dilution of the bacteria of 2-fold,4-fold, 8-fold and 16-fold was carried out

O The sample (A-I) prepared by Frankie wasadded into the wells

O 95μl each and a duplicate was done

O Finally, 5μl of MQAE was added into thewells with the samples

O The microplate was put into the incubator at37°C for 1 hour.

10. Measuredthe MQAE fluorescence by microplate reader.

1 Sept (Thu)

1. Nanodrop

a. PCR amplified HR & T7

Sample	ng/ul	260/280
HR	5.4	1.83
T7	27.1	1.73

b. Ligation

Sample	ng/ul	260/280
HR1C3 @16^oC (1)	16.8	1.76
(2)	18.6	1.76
(3)	12.8	1.79
(4)	16.1	1.78
HR1C3 @22^oC (1)	5.7	1.96
(2)	6.9	1.69
(3)	5.2	1.77
(4)	7.5	1.64

c. Gel recovery

O HR1C3 gel clean: 2.0ng/ul

2. Rungel to check size

O PCRamplified HR & T7 aboth correct

O HR1C3,T71C3awrong size

O HR-T7ano band

3. Restrictioncut: HR, T7, pSB1K3

O HR, T7, 1C3: EcoR1 & Pst1

O HR: Xbal1

O T7: Spe1

4. Ligation

O HR1K3

O T71K3

O HR-T7

O 16^oC, overnight

5. Transformationof gel clean HR1C3 to DH5a

O Spread 3 plates

2 Sept (Fri)

1. Transferlab

2. Pickcolony from plate prepared on 1/9

3. Preparedand autoclaved the following solutions

O 500ml LB solution

O 250ml LB solution with 1M NaCl

O 250ml LB solution with 1M KCl

3 Sept (Sat)

1. Miniprepof DH5a with HR1C3

2. Rungel with miniprep product

Week 14 Monday 5 Sept – Sunday 11 Sept

5 Sept (Mon)

1. Transformationof chloride sensor and 24C terminator

2. Nanodropof HR1C3 prepared on 3/9

sample	ng/ul	260/280
1	46.6	1.85
2	60.5	1.81
3	58.9	1.88

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