Team:Heidelberg/Notebook/Killing I/Notebook/week2

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Week 2

project planning
Guys, I sent an email to Prof. Bagdasarian asking about the RP4 resistance and also if he can send us directly the plasmid and not the strain. Meanwhile, I found this paper:

Distribution of Restriction Enzyme Recognition Sequences on Broad Host Range Plasmid RP4: Molecular and Evolutionary Implications

Brian M. Wilkinsa, f1, Paul M. Chilleya, Angela T. Thomasa and Michael J. Pocklingtona a Department of Genetics, University of Leicester, Leicester, LE1 7RH, England

Please, tomorrow morning check this paper, it should contain the sequence of the RP4 plasmid. In this case, we should be able to find out which resistance is there (if any....).

Good night, Barbara

Strategy for progress:
 * 1) we ordered SanDI (restriction enzyme), GeneRuler 1kb Ladder plus, GeneRuler DNA Ladder High range and a gel extraction kit (QIAEX II) for extraction of large fragments
 * 2) our plan is to doubledigest lambda DNA with XhoI/XbaI, clone this fragment into a vector and amplify it in E.coli. This fragment is than extracted again and digested with SanDI to get an fragment with XhoI and SanDI ends. This fragment includes the necessary GAM site. This fragment is than ligated with our inclusion cassette which has SanDI and XbaI ends (generation described below). This big fragment (ends XhoI and XbaI with oriT and AB cassette) is than ligated in the lambda fragment lacking its XhoI/XbaI fragment. The now complete new lambda phage is than transferred into the killer strain by transformation or in vitro packaging.
 * 3) design of our insert: our insert includes the oriT, the AB cassette (probably Chloramphenicol) and a terminator. All these fragments are separetly amplified by PCR while cloning specific sticky ends to the fragments (oriT: SanDI/BamHI; AB: BamHI/NcoI; terminator: NcoI/XbaI). Our insert finally consists of the oriT, the AB cassette and the terminator. This big fragment has a SanDI and a XbaI end, necessary for the later ligation with the lambda fragment (XhoI/SanDI).

chloramphenicol resistance cassette

 * 1) Mini Prep P1004,1000, B0014, 0015
 * 2) * concentration
 * 3) ** B0014: 14,6ng/ul;
 * 4) ** B00142: 28,4ng/ul;
 * 5) ** B0015: 11,4ng/ul
 * 6) ** P1000: 22,6ng/ul
 * 7) ** P10041: 28,8ng/ul

lambda phage and cI

 * 1) retransformation of mini prep BBa_J01101 into top 10 for maxi prep
 * 2) inoculate for infection test again (see 07.08.08 Do)
 * 3) test digestion of lambda phage
 * 4) * 2ul lambda 857 (600ng), 2ul 10xBSA, 2ul 10xNEBuffer2, 0,5ul XhoI (10U/ul), 13,5ul H2O
 * 5) * incubate 2h
 * 6) *expected fragment lengths: 33498bp and 15003bp
 * 7) * run the gel: 0,5% agarose, 25V, from 20:00 over night

project planning
Prof. Bagdasarian replied to my email:

Dear Barbara: RP4 carries resistances to Ap, Km and Tc. It was easier to send you a strain containing the plasmid since we do not have purified DNA of the plasmid at this time. Please note that the description of the strain we have sent you only indicates Ap. I don't know the reason for that. It would, therefore, be good to check the resistances when you receive the strain.

Michael

So, when we receive the strain we will check the resistances as suggested.

chloramphenicol resistance cassette

 * 1) test digestion of plasmids
 * 2) * P1004 (A,B,C,D,1 and 2) and P1000 with DraI (0,15ul enzyme, 2ul NEB-buffer 4, 10ul DNA per 20ul sample)
 * 3) * B0014 (1 and 2) and B0015 with SfcI (0,3ul enzyme, 2ul NEB-buffer 4, 10ul DNA per 20ul sample)
 * 4) inoculate for 2h at 37°C
 * 5) expected fragments
 * 6) * B0014 and B0015: 1710,824,464,191
 * 7) * P1004
 * 8) * P1000


 * 1) agarose gel of digestion above with very bad results: DNA concentration too low


 * 1) therefore a retransformation done with
 * 2) * P1004-D, P1004-1, P1000, B0015, B0014-1 and B0014-2

cI

 * 1) inoculation of cultures for maxiprep:
 * 2) * cI-retransformation from yesterday: 2 samples in 250ml LB + Amp

cI

 * 1) Maxiprep CI
 * 2) Concentration Measurement of obtained DNA with Nanodrop
 * 3) *Result: two epis with each 100ul DNA (1ug/ul);
 * 4) Restriction digest of obtained CI DNA with SfcI and HindIII (NEB Buffer 4)
 * 5) *Result: High probability that the plasmid is CI, but the image we got is not enough to prove it.


 * 1) results of infection test from 12.08., 5h at 37^C and 15h at RT
 * 2) * MG1655 positiv control, lambda 10^-4 >10000
 * 3) * MG1655 positiv control, lambda 10^-6 1
 * 4) * MG1655 positiv control, lambda 10^-8 9
 * 5) * TOP10 positiv control, lambda 10^-4  3000-5000
 * 6) * TOP10 positiv control, lambda 10^-6  6
 * 7) * MG1655 positiv control, lambda 0     0
 * 8) * TOP10 positiv control, lambda 0      0
 * 9) * MG1655 cI, A           lambda 10^-4  150
 * 10) * MG1655 cI, A           lambda 10^-6  0
 * 11) * MG1655 cI, A           lambda 10^-8  0
 * 12) * MG1655 cI, B           lambda 10^-4  100
 * 13) * MG1655 cI, B           lambda 10^-6  0
 * 14) * TOP10 cI, A            lambda 10^-4  300-500
 * 15) * TOP10 cI, A            lambda 10^-6  0
 * 16) * TOP10 cI, B            lambda 10^-4  0
 * 17) * TOP10 cI, B            lambda 10^-6  0
 * 18) **postitiv control: -cI, -Amp, competent cells


 * some mistakes occured during the experiment --> experiment has to be done again

lambda phage

 * 1) Restriction digest of lambdaDNA (with XhoI) to test new AGE-machine
 * 2) * running over night at 25V/10cm

lambda phage

 * Inoculating of competent MG1655 cells (9:30) (3 x 30µl in 20ml Standard I Medium with Maltose) for Phage production
 * Aliquot of the three populations every half an hour to monitor cell population growth and destruction by the phages (not done)
 * Adding of plaques to the liquid cultures at 13:00
 * Stopping the incubation at 19:00, centrifugation of the cells to pellet (3750rpm,7min), sterile filtration of the supernatant
 * infection test of the supernatant (Yin)

chloramphenicol resistance cassette

 * Miniprep of Antibiotica cassette and terminator and Nanodrop evaluation of obtained DNA with
 * P1000 1 106,1 ng/ul
 * P1000 2 9,6 ng/ul
 * P1004 1 39,5 ng/ul
 * P1004 2 67,2 ng/ul
 * B0014 1 55,2 ng/ul
 * B0014 2 5,9 ng/ul
 * B0015 A1 48,5 ng/ul
 * B0015 A2 31,5 ng/ul
 * B0015 K1 44,2 ng/ul
 * B0015 K2 26,1 ng/ul

project planning

 * New cloning strategy
 * due to the missing of SanDI (not available), our cloning strategy has to be changed
 * digest lambda DNA with XbaI and XhoI
 * digest the small fragment (~9kb) with AgeI
 * discard the fragment with INT/XIS (~7,3kb), the other fragment contains the necessary GEM (~1,7kb)
 * ligate: chloramphenicol cassette, GFP, oriT and the GEM-fragment
 * (chloramphenicol cassette amplified by PCR)
 * we have to check if there are AgeI restriction sites in the DNA fragments to be cloned
 * no AgeI restriction sites could be found --> good
 * results: there is no restriction site of AgeI, XhoI or XbaI in the oriT, GFP and the chloramphenicol sequence

chloramphenicol resistance cassette

 * analytical Digestions of
 * P1000-1, B0014-1, B0015 1 and 2 both grown on K and on A, P1004 1 and 2,
 * preparative digestion of P1000-1
 * results: there was only the undigested vector on the gel - plan to cut the chloramphenicol casette out and to ligate it into another vector abandonded for the moment
 * where lies the problem? Is the right plasmid in the registry, the wrong sequence in the database or did the enzyme fail to work?


 * Sequencing of Miniprep DNA B0015 A1, B0014 1, P1000 1, cI

Saturday, 08/16/08

 * Retransformation of P1000, P1004, B0014, P1000, cI

evaluation of sequencing data

 * cI has not the correct sequence
 * P1000 has not the correct sequence
 * B0014 has not the correct sequence
 * B0015 has the right sequence!