Team:Heidelberg/Notebook/Killing I/Notebook/week3

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   Home    Team   Overview    Advisors  </a></li>  Undergraduates  </a></li>  University  </a></li>  DKFZ  </a></li>  BioQuant  </a></li>  BioRegion Rhein-Neckar  </a></li> </ul> </li>  Project</a> <ul class="DropDownMenu" id="MB1-DDM1">  Overall Project  </a></li>  Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Sensing"> Sensing  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_I"> Killing I  - Phages  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_II"> Killing II - Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Visualization"> Visualization  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Parts" style="color: white">Parts</a> <ul class="DropDownMenu" id="MB1-DDM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts"> Submitted Parts  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts/Characterization"> Characterization  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling</a> <ul class="DropDownMenu" id="MB1-DDM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling"> Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Chemotaxis"> Chemotaxis-Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Phage"> Phage Dynamics model  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook</a> <ul class="DropDownMenu" id="MB1-DDM5"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Sensing >  </a> <ul class="SideMenu" id="MB1-DDM2-SM1"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Killing I - Phages >  </a> <ul class="SideMenu" id="MB1-DDM2-SM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Killing II - Colicin >  </a> <ul class="SideMenu" id="MB1-DDM2-SM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/visualization"> Visualization  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/material"> Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/team_meetings"> Team Meetings  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/seminar"> Seminar on Synthetic Biology  </a> </ul> </li> <li style="width: 160px"> <a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice</a> <ul class="DropDownMenu" id="MB1-DDM4"> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview"> Project Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Phips_the_Phage"> Phips the Phage  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Essay"> Essay  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"> Surveys  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"> Open Day  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"> Nobel Prize  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Sponsors" style="color: white">Sponsors</a> </li> </ul>

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Week 3

chloramphenicol resistance cassette
2µl DNA 5µl NEB2 5µl BSA 1µl XbaI 1µl XhoI 26µl water
 * Maxiprep of P1000, P1004, B0014, B0015
 * P1000 1672 ng/µl 1,92
 * P1004 1153 ng/µl 1,92
 * B0014 2040,4 ng/µl 1,92
 * B0015 1053,6 ng/µl 1,92
 * Analytical digestions
 * lambda DNA with XbaI and XhoI

1µl DNA 2µl NEB3 2µl NcoI 15µl water
 * Analytical digestion of P1000

1µl DNA 2µl NEB4 1,5µl DraI 15,5µl water
 * Analytical digestion of P1004

1µl DNA 2µl NEB4 2µl BSA 2,5µl SfcI 12,5µl water
 * Analytical digestion of B0014 / B0015


 * Analytical digestion of cI "green", cI "black" and T9002


 * Gel
 * 1) * Lane 0: DNA ladder mix
 * 2) * Lane 1: lambda DNA
 * 3) * Lane 2: lambda DNA (XbaI, XhoI --> large frament ~ 40kb, small fragment ~ 9kb)
 * 4) * Lane 3: P1000
 * 5) * Lane 4: P1000 (NcoI-->1473,2374)
 * 6) * Lane 5: P1004
 * 7) * Lane 6: P1004 (DraI-->19,339,692,1067,1360)
 * 8) * Lane 7: cI green
 * 9) * Lane 8: cI green (NdeI - expected fragments?)
 * 10) * Lane 9: cI black
 * 11) * Lane 10: cI black (NdeI)
 * 12) * Lane 11: T9002
 * 13) * Lane 12: T9002 (NdeI - expected fragments?)

lamda phage
Infectiontest
 * inoculation of 20ml medium with over night culture (MG1655)
 * after 1h plaque added --> 37C for 2h + inoculation of ZMBH phage (100ql 10^-2 in 20ml MG1655)
 * incubation at 42C for 2 more hours
 * sterilefiltration + mix with growing bacteria (MG1655 and MG1655+cI)
 * incubate in soft agar at 37C for 2h, than at 42C for another 4h (altogether 24 plates)
 * phage DNA was also isolated from filtrate
 * results:
 * same amount of plaques on plates with MG1655 as with MG1655+cI
 * nearly no plaques on plates with own phage --> very low phage concentration
 * --> cI does not work!!!!
 * growing curve
 * the values are the average of all four cultures

lambda phage

 * Digestion of lambda DNA with XbaI and XhoI
 * very nice separation of the small fragment from the two large fragments (one band) picture of the gel was not saved but it looked very nice
 * cutting out of the small and large fragment --> gel purification kit
 * small fragment: 30,3 ng/µl; 1,86
 * large fragment1: 19,5 ng/µl; 2,11
 * large fragment2: 61,5ng/µl; 1,90

GFP

 * inoculation of three overnight cultures from the two I20260 glycerol stocks

chloramphenicol resistance cassette

 * Transformation of P1000, P1004 (both chloramphenicol resistances) and CI
 * no success

project planning
Guys, we received this email by another professor in the states. sounds good. we should get this helper plasmids in the end :)

I have sent pUB307, RSF1010, pED350, pED361, which are described in our two MGG papers. Also two clones I made of the oriT from RP1; pED369 and pED374. There are 10microl of each. The DNAs are old (1983!) and so I would transform and make fresh plasmid preps.

pED350 is oriT+ and Mob+, so it is efficiently mobilised by RPI or its derivative pUB307. pED361 is oriT+ only. It cannot be mobilised by pUB307 as it lacks mob functions. If RSF1010 is also in the cell pKD361 is very efficiently mobilised by pUB307.

pED369 and pED374 are two subclones I made of the RP1 oriT into pED825 (Amp resistant described in MGG papers). pED374 contains a single 690 bp HaeII fragment containing the RP1 oriT. PED369 contains the 690 bp oriT fragment plus additional HaeII fragments. I sent both just in case the pED374 DNA does not check out. These are efficiently mobilised by pUB307.

I would think cloning this 690 bp fragment into your lambda vector would be the simplest solution; its small and will mobilise in the presence of just pUB307.

Keith

GFP

 * Miniprep of reference promotor I20260
 * Concentrations:
 * Epi1 9,8 ng/ul; 2,52
 * Epi2 15,7 ng/ul; 2,28
 * Epi3 19,7 ng/ul; 1,93

10 µl DNA (from Epi3) 2ul SmlI 12µl NEB4 2ul BSA 10x 4 µl water
 * Digestion of I20260

18 µl DNA (from gel purification kit) 5 µl AgeI 5 µl NEB4 22 µl water
 * Digestion of the XbaI-XhoI fragment with AgeI


 * Gel




 * Lane 0: DNA ladder mix
 * Lane 1: I20260 (SmlI-->251bp, 373bp, 843bp, 918bp, 1284bp)
 * Lane 2: XbaI-XhoI fragment (AgeI-->1.7kb, 7.3kb)
 * Lane 3: large lambda DNA fragments 3µl (15kb, 24.5kb)


 * Results:
 * I20260 seems not to be correct, inoculate overnight cultures from the glycerol stocks again
 * digestion of the XbaI-XhoI fragment with AgeI did not work good, only the 7.3kb fragment can be seen, reasons: wrong buffer concentration, too much enzyme (glycerol) in the reaction
 * large lambda DNA fragment is looking good

lambda phage

 * concentrations of lambda-DNA extractions
 * from Fermentas Phage - plaque 1: 7.5 ng/ul; 1.86
 * from Fermentas Phage - plaque 2: 61.5 ng/ul 1.90
 * from ZMBH phage 19,7 ng/ul 2,11

overview of the phage cloning strategy one


Used primer sequences:

oriT_RP4_fw (Tm=62°C):

CTCGTTTCTAGAACTAGTgacaggctcatgccggccgc

oriT_RP4_rv: (Tm=58°C)

TATTCGGGTACCgtcccctcagttcagtaatttcctgc

GFP_CmR_fw: (Tm=56°C)

CTCGTTGGTACCTCTAGAtttacagctagctcagtcctagg

GFP_CmR_rv: (Tm=55°C)

TATTCGACCGGTACTAGTtataaacgcagaaaggcccacc

GAM_fw: (Tm=55°C)

AGTGCTTTAGCGTTAACTTCCG

GAM_rv: (Tm=53°C)

GGTTTTACCGCATACCAATAACG

CmR_fw: (Tm=54°C)

gctaaaATGgagaaaaaaatcactgg

CmR_rv: (Tm=58°C)

AGGTTCTCCTTTATTAGCCGGATCCTCTAGATTACGCC

lambda phae
10µl DNA = 3 ug 2µl XhoI 2µl XbaI 5µl NEB 2 5µl BSA 33µl H20 36 µl DNA 2 µl XhoI 2µl XbaI 5µl NEB 2 5 µl BSA
 * Preparative digestion of lambda-DNA from Fermentas
 * lambda DNA "2" and "ZMBH" from phage extraction kit
 * all digestions 3h, 400rpm, 37°''


 * Gel
 * lane 0: DNA ladder mix
 * lane 2: lambda DNA (fermentas) (XbaI, XhoI --> ~40 kb, ~9 kb)
 * lane 4: lambda DNA from extraction "2" (XbaI, XhoI --> ~40 kb, ~9 kb)
 * lane 6: lambda DNA from extraction "ZMBH phage" (XbaI, XhoI --> ~40 kb, ~9 kb)
 * lane 2, 4 and 6 overflowed
 * cutting out of the XbaI-XhoI fragment from lane 2
 * the large fragment wasn't cutted out because the gel was exposed to UV light over 30 seconds
 * gel extraction kit
 * DNA eluted in 20µl

20µl DNA (from gel purification kit) 2µl AgeI 5µl NEB4 23µl water
 * Digestion of the XbaI-XhoI fragment with AgeI
 * 16°C over night

10µl DNA = 3 µg 2µl XhoI 2µl XbaI 5µl NEB 2 5µl BSA 33µl water
 * Preparative digestion of lambda-DNA from Fermentas
 * 16°C over night

GFP

 * Miniprep from overnight cultures of I20260 glycerol stocks
 * eluted in 50 µl
 * Concentrations:
 * Miniprep 1: 19.8 ng/µl
 * Miniprep 2: 17.4 ng/µl
 * Miniprep 3: 19.0 ng/µl
 * Miniprep 4: 16.4 ng/µl


 * Analytical digestion of the four I20260 Minipreps
 * 20µl DNA
 * 5µl NEB 4
 * 1.5µl DraI
 * 23.5µl water


 * Gel
 * lane 0: DNA ladder
 * lane 1: I20260 1
 * lane 2: I20260 1 (DraI-->19bp, 535bp, 886bp, 2229bp)
 * lane 3: I20260 2
 * lane 4: I20260 2 (DraI)
 * lane 5: I20260 3
 * lane 6: I20260 3 (DraI)
 * lane 7: I20260 4 (DraI)


 * Results
 * it seems that we have I20260, inoculation of an overnight culture from I2020 No. 3 for Maxiprep


 * Transformation of I20260 (Kan) (from the promotor measurement kit sheet as well as from the registry) and J01101 (Amp)
 * eluted in 10µl water
 * transformed into TOP10 and MG1655 (each 4µl DNA)

cI

 * ordered J01101 (cI) from the iGEM headquater, they will already send it today

GFP

 * Maxiprep of reference promotor I20260
 * concentrations: 200 ng/µl

lambda phage

 * Gel: Digestions of lambda-DNA (fermentas), and the small XbaI-XhoI fragments




 * lane 1: DNA ladder mix
 * lane 3: lambda-DNA (XbaI, XhoI; ~ 40kb, ~ 9kb)
 * lane 5: small lambda-fragment assayI (AgeI, ~1,7kb, ~7,3kb)
 * lane 7: small lambda-fragment assayII (AgeI, ~1,7kb, ~7,3kb)


 * Extraction of XbaI-XhoI fragments (evaluation of samples with nanodrop)
 * small fragment 1: 15,9 ng/µl; 2,41
 * small fragment 2: 6,6 ng/µl; 2,02
 * large fragment 1: 14,1 ng/µl; 2,75
 * large fragment 1: 10,8 ng/µl; 2,86
 * large fragment 1: 14,4 ng/µl; 1,99
 * large fragment 1: 16,8 ng/µl; 2,14