Team:Heidelberg/Notebook/Killing I/Notebook/week12

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Week 12

Monday, 10/20/08

Proceedings of phage cloning strategy one

the fully mutated insert in pBluescript SK II was digested with XbaI/XhoI and the insert extracted from the gel
lambda DNA was also cut with XbaI/XhoI and the backbone also extracted from the gel.
the insert was ligated in the lambda backbone over night using standard ligation methods

Phage cloning strategy two

Digestion with XbaI/XhoI
- should be: 3945, 2898
Gel

lane 1-8: new minipreps
lane 9: old insert fully mutated
results: the new minipreps seem to be the old unmutated pBluescript

Characterization of oriT

Quantitatively test for oriT

Donor: overnight culture Top10 oriT+pUB307 OD(600nm): 1.548
Recipient:
overnight culture Top10 pBAD 33 OD(600nm): 1.808
overnight culture MG1655 pBAD 33 OD(600nm): 2.932
overnight culture DH5alpha pBAD 33 OD(600nm): 1.976

- Centrifuge overnight culture in 1.5ml eppi for 2min at 13000rpm, 24 samples donor, 510ul/sample; 8samples Top10 recipient, 450ul/sample; 8samples MG1655 recipient, 375ul/sample; 8samples DH5alpha recipient, 600ul/sample;
- Wash the pellet twice with LB medium
- Resolve the pellet in LB medium
- Centrifuge the washed recipient for 2min at 13000rpm, discard the fluid
- Add the washed donor suspension
- Vortex and resolve the pellet
- Centrifuge the mix for 1min at 13000rpm
- Resolve the pellet in 100ul LB
- Put membrane filter on the LB agar
- Pipett the suspension on membrane filter (8samples*3tests)
- Incubate the plates with membrane filter at 37°C
- Put directly one membrane filter into 1ml LB in an 1.5ml eppi
- Vortex the eppi for 30sec, dilute for 10-5 and 10-6, plate them out on LB/Amp+Cm plates (0min)
- For Top10 as recipient: plate them out also on LB/Kan+Cm plates and LB/Amp+Kan+Cm plates
- After 6, 12, 18, 24, 30, 36, 42 min repeat the last three steps.
- Negative control plates:
  - LB/Cm+Amp:

100ul donor overnight culture
100ul recipient overnight culture (x3)

- Cell number determination
  - LB/Cm: 100ul 10-6 recipient overnight culture (x3)
  - LB/Kan+Amp: 100ul 10-6 donor overnight culture

Result:
- Negative control: negative
- Colony on LB/Cm:

Top10:166 (Titer of recipient: 1.66e9/ml)
MG1655:194 (Titer of recipient: 1.94e9/ml)
DH5alpha:115 (Titer of recipient: 1.15e9/ml)

- Colony on LB/Kan+Amp: 140 (Titer of donor: 1.4e9/ml)

(information for volume counting: 1OD Top10 = 9e8/ml; 1OD MG1655 = 6.6e8/ml; 1OD DH5alpha = 5.8e8/ml)

- Colony on other LB/Cm+Amp plates, LB/Kan+Cm plates and LB/Amp+Kan+Cm plates:
  - 10-5 dilute:

Time	Top10-CA	Top10-CK	Top10-CKA	MG1655
0	2	1	0	7
6	17	4	1	22
12	194	90	7	480
18	230	109	49	1180*
24	1130*	800*	136	1920*
30	2500*	1540*	260	2960*
36	2600*	1800*	920*	3440*
42			960*	4040*

'*':10-6 dilute

Tuesday, 10/21/08

Proceedings of phage cloning strategy one

the emerging plasmid consisting of our insert in tha lambda phage was transformed in E. coli by chemical transformation and in vitro packaging
the bacterias were plated on chloramphenicol and incubated at 28 °C to ensure that the thermolabile cI (cI857) was not degraded

Phage cloning strategy two

do again a digestion of mutagenesis pcr minipreps from 10/07/08 but with an additional restriction enzyme (EcoRI)to get a better separation on gel to cut out the backbone
digestion with KpnI/AgeI/EcoRI
- mutagenesis pcr succesful: 300bp, 1.4kb 5.1kb
- mutagenesis pcr not succesful: 300bp, 1.4kb, 1.7kb, 3.4kb

-->mutagenesis pcr on the first three lanes successful
- cut out 5.1kb band and gel purification kit

lane0: DNA ladder mix
lane1: purified 5.1kb band from last gel (pBluescript backbone from phage cloning strategy one (KpnI/AgeI)
lane2: CmR1 digested (KpnI/SacI)
lane3: CmR2 digested (KpnI/SacI)
lane4: GFP1 digested (SacI/AgeI)
lane5: GFP2 digested (SacI/AgeI)

ligation of purified 5.1kb band from last gel (pBluescript backbone from phage cloning strategy one (KpnI/AgeI) with CmR1/2 and GFP1/2
- 30min at room temperature
transformation in TOP10

Characterization of oriT

Inoculate the cells for conjugation test

15ml LB/chloramphenicol + glycerol stock Top10 pBAD 33 (x2)
15ml LB/kanamycin/ampicilin +glycerol stock Top10 oriT+pUB307 (x2)

Wednesday, 10/22/08

Proceedings of phage cloning strategy one

first small colonies can be seen on chloramphenicol plates.
colonies were picked and transferred in liquid media containing chloramphenicol

Characterization of oriT

Qquantitatively test for oriT (twice)

Donor 1: overnight culture Top10 oriT+pUB307 OD(600nm): 2.828
Donor 2: overnight culture Top10 oriT+pUB307 OD(600nm): 3.004
Recipient 1: overnight culture Top10 pBAD 33 OD(600nm): 3.116
Recipient 2: overnight culture Top10 pBAD 33 OD(600nm): 3.084

- Centrifuge overnight culture in 1.5ml eppi for 2min at 13000rpm, 15samples donor1, 15samples recipient1, in different volume (see the result table below)
- Wash the pellet twice with LB medium
- Resolve the pellet in LB medium
- Centrifuge the washed recipient for 2min at 13000rpm, discard the fluid
- Add the washed donor suspension
- Vortex and resolve the pellet
- Centrifuge the mix for 1min at 13000rpm
- Resolve the pellet in 36ul LB
- Put membrane filter on the LB agar
- Pipett the suspension on membrane filter (15samples)
- Incubate the plates with membrane filter at 37°C for 20min
- Put membrane filter into 1ml LB in an 1.5ml eppi
- Vortex the eppi for 30sec, dilute for 10-6, plate them out on LB/Amp+Cm plates
- Negative control plates:
  - LB/Cm+Amp:

100ul donor 1 overnight culture
100ul recipient 1 overnight culture

- Cell number determination
  - LB/Cm: 100ul 10-6 recipient 1 overnight culture
  - LB/Kan+Amp: 100ul 10-6 donor 1 overnight culture

Repeat the test with donor 2 and recipient 2.

Result:
- Negative control: negative
- Colony on LB/Cm:

recipient 1: 458 (Titer of recipient: 4.58e9/ml)
recipient 2: 403 (Titer of recipient: 4.03e9/ml)
Colony on LB/Kan+Amp:
donor 1: 347 (Titer of donor: 3.47e9/ml)
donor 2: 288 (Titer of donor: 2.88e9/ml)

- Reaction volume and colony on other LB/Cm+Amp plates:

Sample	Donor 1(ul)	Rezipient 1(ul)	colony 1	Donor 2(ul)	Rezipient 2 (ul)	colony 2
50:1	289	5.1	12	289	5,2	16
25:1	283	10	28	283	10.1	31
10:1	268	24	62	253	24	63
8:1	262	29	81	247	29	75
5:1	246	44	115	231	44	120
3:1	221	66	161	208	66	129
2:1	196	88	213	185	88	212
1:1	147	132	302	139	132	303
1:2	98	175	351	93	175	298
1:3	74	197	294	69	197	302
1:5	49	219	289	46	219	194
1:8	35	234	241	31	234	190
1:10	27	239	158	25	239	134
1:25	11.3	253	86	10.7	253	82
1:50	5.8	258	31	5.4	258	30

Inoculate the cells for conjugation test

15ml LB/chloramphenicol + glycerol stock Top10 pBAD 33 (x2)
15ml LB/kanamycin/ampicilin +glycerol stock Top10 oriT+pUB307 (x2)

Thursday, 10/23/08

Proceedings of phage cloning strategy one

colonies could grow in chloramphenicol containing medium --> samll aliquots were transferred in ampicillin containing medium to check for the resisitance
colony PCRs were conducted to ensure that our insert is embedded in the growing cells
pUB307 was transferred in cells harboring our modified lambda phage by conjugation --> plating on Cm + Kan

phage cloning strategy two

inoculation of liquid cultures from ligation (tuesday)

Characterization of oriT

Quantitatively test for oriT (twice)

Donor 1: overnight culture Top10 oriT+pUB307 OD(600nm): 1.404
Donor 2: overnight culture Top10 oriT+pUB307 OD(600nm): 1.288
Recipient 1: overnight culture Top10 pBAD 33 OD(600nm): 1.428
Recipient 2: overnight culture Top10 pBAD 33 OD(600nm): 1.292

- Centrifuge overnight culture in 1.5ml eppi for 2min at 13000rpm, 15samples donor1, 280ul/sample; 15samples recipient1, 530ul/sample;
- Wash the pellet twice with LB medium
- Resolve the pellet in LB medium
- Centrifuge the washed recipient for 2min at 13000rpm, discard the fluid
- Add the washed donor suspension
- Vortex and resolve the pellet
- Centrifuge the mix for 1min at 13000rpm
- Resolve the pellet in 40ul LB
- Put membrane filter on the LB agar
- Pipett the suspension on membrane filter (15samples)
- Incubate plates with membrane filter at different temperature for different time

	4°C	22°C	30°C	37°C	42°C
20min	1plate	1plate	1plate	1plate	1plate
40min	1plate	1plate	1plate	1plate	1plate
60min	1plate	1plate	1plate	1plate	1plate

- Put membrane filter after the incubation into 1ml LB in an 1.5ml eppi
- Vortex the eppi for 30sec, dilute for 10-6, plate them out on LB/Amp+Cm plates
- Negative control plates:
  - LB/Cm+Amp:

100ul donor 1 overnight culture
100ul recipient 1 overnight culture

- Cell number determination
  - LB/Cm: 100ul 10-6 recipient 1 overnight culture
  - LB/Kan+Amp: 100ul 10-6 donor 1 overnight culture

Repeat the test with donor 2 (300ul/sample) and recipient 2 (590ul/sample).

Result:
- Negative control: negative
- Colony on LB/Cm:

recipient 1: 138 (Titer of recipient: 1.38e9/ml)
recipient 2: 99 (Titer of recipient: 0.99e9/ml)

- Colony on LB/Kan+Amp:

donor 1: 129 (Titer of donor: 1.29e9/ml)
donor 2: 110 (Titer of donor: 1.1e9/ml)

- Colony on other LB/Cm+Amp plates:
  - Donor 1 + recipient 1

Temperatur 20min 40min 60min
4°C 5 5 1
22°C 2 78 117
30°C 71 178 241
37°C 154 285 550
42°C 167 273 402

- - Donor 2 + recipient 2

Temperatur 20min 40min 60min
4°C 2 1 0
22°C 29 61 79
30°C 43 117 227
37°C 118 290 332
42°C 122 271 234

Friday, 10/24/08

Proceedings of phage cloning strategy one

colonies grew on plates containing chloramphenicol and kanamycin --> were picked and liquid medium inoculated
conjugation of our lambda phage into cells harboring T9002 in pSB1A3 --> brought out on plates containing chloramphenicol and ampicillin

phage cloning strategy two

Miniprep of overnight cultures
4 Control digestions to check wheter the ligation was successful or not

lane0: DNA ladder mix
lane1: XbaI/XhoI: 3,9kb / 2,9kb (NEB2, BSA)
lane2: DraI: 1,85kb / 1,8kb / 1,35kb / 780bp / 692bp/ 339bp/ 19bp (NEB4)
lane3: SacI/SpeI: 4.5kb / 1.3kb / 929bp / 34bp (NEB1,BSA)
lane4: SfcI: 3.1kb / 1.3kb / 1.1kb / 678bp / 247bp / 192bp (NEB4, BSA)

we got the correct pBluescript with the insert of phage cloning strategy two

Saturday, 10/25/08

Proceedings of phage cloning strategy one

we got colonies on the plates with ampicillin and chloramphenicol only when our conjugated cultures were brought out, but not if T9002 or the cells harboring pUB307 and our phage were brought out separately
this shows that our phage can be transported by conjugation

Sunday, 10/26/08

Chloramphenicol resistance cassette

Experiment, to check until what antibiotic concentration the plasmid gives resistance
inoculation of TOP10 and TOP10 with BBa_K150003 in LB with different Cm concentrations:
- 0 µg/ml
- 5 µg/ml
- 10 µg/ml
- 15 µg/ml
- 20 µg/ml
- 25 µg/ml
- 30 µg/ml
- 35 µg/ml
- 40 µg/ml
- 45 µg/ml
- 50 µg/ml
- 60 µg/ml
- 70 µg/ml
- 80 µg/ml
- 90 µg/ml
- 100 µg/ml
- 250 µg/ml
- 500 µg/ml
- 750 µg/ml
- 1000 µg/ml
- 5000 µg/ml
stock solution: 100mg/ml in ethanol
mock treated:
- 500 µg/ml
- 750 µg/ml
- 1000 µg/ml
- 5000 µg/ml

meausurement of OD every 30min with TECAN, measurement length: 12 hours

<< Week 11	Overview	Week 13 >>

Team:Heidelberg/Notebook/Killing I/Notebook/week12

From 2008.igem.org

Contents

Monday, 10/20/08

Proceedings of phage cloning strategy one

Phage cloning strategy two

Characterization of oriT

Tuesday, 10/21/08

Proceedings of phage cloning strategy one

Phage cloning strategy two

Characterization of oriT

Wednesday, 10/22/08

Proceedings of phage cloning strategy one

Characterization of oriT

Thursday, 10/23/08

Proceedings of phage cloning strategy one

phage cloning strategy two

Characterization of oriT

Friday, 10/24/08

Proceedings of phage cloning strategy one

phage cloning strategy two

Saturday, 10/25/08

Proceedings of phage cloning strategy one

Sunday, 10/26/08

Chloramphenicol resistance cassette