Team:Heidelberg/Notebook/Killing I/Notebook/week1

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Latest revision as of 11:31, 29 October 2008

Week 1

Monday, 08/04/08

ZMBH lambda phage

• infection test
  • Bacteria: Top10, DH5alpha, 10^8/softagar
  • Lambda-Phage: Lambda cI- from ZMBH practical lab, A.10^7/softagar; B.10^5/softagar;
  • Incubate: 37°C
  • Results:
    • after 5h – no plaques
    • after 7h – no plaques
    • after 20h – DH5alpha:
      10^7 Phage – complete lysed
      10^5 Phage – 500~1000 plaques
      Top10: both – 50~150 plaques
      
      

• transformation: BBa_J01101 (pTet-StrRBS-cI-DbITerm)from the registry (1004 10F) into Top10.
• Result: colony: yes. Inoculate: 4 (A/B/C/D)

• Miniprep of BBa_J01101
• phage buffer (not finished, wait for gelatine, already booked)
• Standard I agar 500ml prepared
  
  

• infection test
  • Bacteria: Top10, Top10:: BBa_J01101 (cI) (Amp resistent), 10^8/softagar
  • Lambda-Phage: Lambda cI- from ZMBH practical lab, 1^7/softagar;
  • Incubate: 37°C 19:00~9:00+
  • Result:
    • TOP10 -Amp +lcI 260 Plaques
    • TOP10 +Amp +lcI viele einzelne Kolonien, kein Rasen, dadurch keine Plaques sichtbar, Kolonien könnten von Kontamination stammen oder von Amp-resistenten TOP10
    • TOP10 +cI +Amp +lcI A:26, B:27, C:12, D:18 Plaques
    • TOP10 +cI -Amp +lcI A:33, B:45, C:38, D:4 Plaques
    • TOP10 +cI -Amp -lcI A:0 , B:0 , C:0 , D:- Plaques
      
      

project planning

1. cloning design: plasmid with constitutive promoter (Barbara) :
   • pBR322(Pbla promotor)? Wait for answer of orther research group
   • restriction sites: no found (alternative: PCR?)
   • New plasmid with cI are found (promotor?)
     
     
2. technics checking: on work
3. First Group Meeting

• Team: Barbara, Jens, Anna, Chistian, Maximilian, Stephen, Yin
• Work in the follow days:
  1. check the MoBi technics to handle lambda phage
     1. extract DNA from the packed virus (produce a easy protocoll <lyse capsid>? Kit?)
     2. argarose gel not seemly for lambda with over 35kb (alternative methods?)
     3. alternative for mini-prep (Kit?)
     4. cloning protocoll?
     5. in vitro packing and transduction (Kit?) or electroporation?
  2. get the help plasmid (wait and write)
  3. testdigestions of lambda cI- phage from ZMBH practical lab (and try to get map from Dr.Mayer?)
  4. check if there are some lambda phage that we can buy (cI-mutant / xis, int deleted)

Tuesday, 08/05/08

project planning

• lambda DNA cI857 from Fermentas ordered (heat inducible cI promotor)
• Quigen lambda-extraction kit ordered

• experimental procedure:
  • test methods for AGE with lambda phage DNA
  • transformation of Phagen-DNA - testing, selection through swaping temperature, infection needs maltose
  • create CI Plasmid with constitutive promotor
  • OriT and AB-Kasette (if possible from the registry) put in a plasmid, therefore we need to know which helper plasmid to use, so we know which oriT to use
  • testconjugation with the helperplasmid - (how to test it - GFP or AB-resistence?)
  • put part 3 in phage vector
  • Sensing Part - control a Relaxase gene through AutoinducerI and knock it out on the helper plasmid

• Parts for the Registry:
  • cI with a constitutive promotor
  • AB-Kasette with promotor
  • lambda DNA ?

• Problems
  • Which OriT, and therefore we have to know which helper plasmid we use?
  • we would use 3 plasmids in the end, so 3 differen antibiotic resistances - the one of the helper plasmid cannot be influenced by us
  • look for suitable restriction sites in the lambda genome
  • selection when using in vitro packaging
  • host range of the phage

Wednesday, 08/06/08

1. Transformation for pTet promotor measurement
   • 2xReference promotor I20260 into Top10, LB/Kan
   • 2xReference promotor I20260 into MG1566 (the killer strain), LB/Kan

cI

1. Transformation BBa_J01101 Mini-Prep (C;D) 3µl into 100µl competent cells
   • 2x into Top10, LB/Amp
   • 2x into MG1566, LB/Amp

lambda phage

1. Transformation Lambda phage cI857(bought) (no selection) 2µl(0,6µg)phage into 50µl bacteria
   • 2x into Top10
   • 2x into MG1566
   • 2x into DH5alpha
   • instead 37C 1h in 400µl LB : 1x 37°C incubate in 400µl LB 15min, 1x 30°C 15min. The plates: 1x 37°C, 1x°30C (6.floor)
   • all: Standard I agar with glucose (plan:when they have grown and overlay the plates, then incubate them at 37°C or 40°C.By positive transformation: we will see the plaques.)

chloramphenicol resistance cassette

1. Transformation of chloramphenicol cassette for lambda phage
   • P1004 (registry 1003:2D), into Top10, LB/Amp, Amp resistence
   • P1000 (registry 1020:11D), into top10, LB/Amp, Amp/Chl resistence
2. Transformation of terminator for chloramphenicol resistence cassette
   • B0015 (registry 1016:5F), into Top10, LB/Amp, LB/Kan
   • B0014 (registry 1016:2E), into Top10, LB/Amp, LB/Kan

Thursday, 08/07/08

• I20260 transformed top10 and MG for promotor mesurement

chloramphenicol resistance cassette

• inoculation of B0014 (Kan),B0015 (Kan),P1004
  • no colony by P1000,B0014 (Amp), B0015 (Amp)

cI

• cI transformed top10 and MG for infection test
• digestion the mini prep BBa_J01101 C and D with HindIII and Scf I
  • Result: not enough DNA

project planning

Hi all, we finally received several answers for the conjugation plasmids. Prof. Erich Lanka, who is now retired, will look for pUB307 in his stocks in Berlin and will contact me again once he has it. I am thinking of asking for his scientific advice, since he also worked with phages... We also received an answer from two professors in the states (actually also another one, who said she did not have pUB307). I copy here the email of one of these, since it gives us something to think about:

Dear Barbara: I do not have the plasmid pUB307. We did, in the past, use helpers to transfer plasmids that have an oriT by conjugation. I am not sure I understand what exactly you wanted to do. do you already have the lambda with an oriT inserted? If yes, which oriT do you have there? If it is the oriT of RSF1010 then the best helper plasmid for its transfer would be a derivative of RP4 (same as RK2). There are strains that have this plasmid integrated in the chromosome so they do not transfer the helper, just the mobilizable plasmid. If you need the RSF1010 oriT I and the strain containing the integrated RP4 we can send these to you. Please note, however, that the "oriT" is not just the transfer origin. It requires two or three genes that are located near the origin and that specify proteins required to attach to the oriT and perform a strand-specific and site-specific nick. One would have to insert this whole fragment into the lambda to make it mobilizable. If you have any questions on this, please do not hesitate to write.

Michael

I will reply explaining better our plans, hopefully he has some good ideas :)

Keep up with the good work, hopefully soon we will have the plasmids to try the conjugation!

Barbara

Friday, 08/08/08

chloramphenicol resistance cassette

1. Mini Prep P1004 (B0014 B0015 no growth)
2. transformation for p1004,1000, B0014,0015 again
   • result: colonies exist, inoculate on sunday

lambda phage and cI

1. infection test on StI Maltose Soft Plattes
   • Top10+Lambda 10^7
   • MG+Lambda 10^7
   • MG+Lambda 10^5
   • Result incubate 37C 18h Platte not on Head, so wat!!
     • Top10 complett lysed
     • Top10 cI complett lysed (not good)
     • MG Lambda 10^7 complett lysed
     • MG cI Lambda 10^7 complett lysed with very big colonies
     • MG cI Lambda 10^5 plaques
     • MG Lambda 10^5 plaques, more than MG cI
   • problems
     • plattes were wat
     • soft agar was sometimes not hart enough (mix!!!)
     • maltose makes the infectionrate much higher - Lambda concentration should be reduced
2. concentration of the cI mini prep
   • C:36,244ng/ul; D:18,035ng/ul;
3. digestion the mini prep BBa_J01101 C and D with HindIII and Scf I
   • result: not good

1. ligation and transformation of lambda 857
   • 2ul lambda (600ng),2ul 10xligation buffer, 1ul DNA T4ligase, 15ulH2O, at RT ca.60min, transform 10ul into 100ul cells(Top10, MG), 30C 18h, 42C 24h
   • result:0 plaque by top10, 5 plaques by MG

project planning

Guys, Prof. Bagdasarian replied:

Dear Barbara: I don't know whether this particular integrated RP4 may be excised from the chromosome and whether it will function as autonomous plasmid. We will send you, on Monday, a strain that carries an autonomous RP4 and a strain carrying the chromosomal RP4 and a small plasmid that contains the "minimal functional oriT" inserted into that plasmid as a NdeI fragment. This derivative is mobilizable at high frequency from that strain due to the "help" of the integrated RP4. You can use either of the strains to PCR-amplify the oriT and, if necessary, add desired sequences with restriction sites to it. Good luck,

Michael

So next week we will hopefully receive the stuff we need to go on with out project :) I hope the ligation works to get lambda into E. coli... Keep me updated, as always.

Ciao, Barbara

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