Team:Freiburg Bioware/NoteBook/Labjournal/October

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Contents

136. labday 01.10.2010

Test Digestions of yesterdays minipreps

Investigator: Achim, Anna

  • 001_CMV: Cut with Eco, Pst & Pvu
    • P654 (Lane 1)
    • P655 (Lane 2)
    • P686 (Lane 3)
    • Control P320 (Lane 4)
  • CMV_affi_VP2/3_capins-HSPG_KO: Cut with Eco & Ssp
    • P680 (Lane 8)
    • P681 (Lane 9)
    • P684 (Lane 10)
    • P685 (Lane 11)
    • Control P613 (Lane 12)
  • CMV_affii_VP2/3_capins: Cut with Eco & Ssp
    • P678 (Lane
    • P679
    • Control P514
  • pCerulean_Vp1up_NLS_Darpin: Cut with BamHI & XbaI
    • P676 (Lane 13)
    • P677 (Lane 14)
    • P683 (Lane 15)
    • Control P365 (Lane 20)
      • Results:The expected bands should run at 500 and 4500 bp. However, just one band at ~3000 bp is visible on the gel.

Samples Stefan: Cut with Xba & Spe

    • P670 (Lane 16)
    • P671 (Lane 17)
    • P672 (Lane 18)
    • P673 (Lane 19)




Freiburg10 01102010 testverdau.png


mini preps of CD clones

Investigator: Kira

c(p689)= 115 ng/ul
c(690)= 151, 20 ng/ul
c(691)= 122,27 ng/ul
c(p692) = 126,42 ng/ul

test digestion of CD clones

Investigator: Kira

Components sample Volume/µL
DNA 4,0 µl
BSA (10x) 2 µl
Buffer no. 4 2,0 µl
Enzyme 1 XbaI 1,0 µl
Enzyme 2 AgeI 1,5 µl
H2O 9,5 µl
Total volume 20


incubation @ 37 C for approx. 2 h

1% agarose gel

Freiburg10 CD testdigestion 2010-10-01.jpg

Biobrick assembly pSB1C3_lITR_hTERT_beta-globin_CD

Investigator: Kira

c(pSB1C3_lITR_hTERT_beta-globin)= 333 ng/ul
c(pSB1C3_CD)= 151 ng/ul

Components vector Volume/µL insert Volume/µL
DNA 4,5 µl 6
BSA (10x) 3 µl 3
Buffer no. 4 3,0 µl 3
Enzyme 1 XbaI 0 µl1,5
Enzyme 2 SpeI 1,5 µl 0
Enzyme 3 PstI-HF 1,0 1
H2O 17 15,5
Total volume 25


incubation @ 37 C for approx. 2 h

1% agarose gel

Freiburg10 CD-beta-globin.jpg

Ligation

DNA-mix: 8 ul (vector 4,6ul)+(insert 3,4 ul)
T4 ligase: 1 ul
T4 buffer: 1 ul
Incubation @ RT for 30 min

Transformation was performed according to the standard protocol w BL21 cells.


Sequencing results of pCerulean_Zegfr:1907_MiddleLinker_VP2/3_HSPG-KO

Investigator: Hanna

Comment: All N-terminal fusion approaches with VP2/3_HSPG-KO revealed positive results except of pCerulean_Zegfr:1907_MiddleLinker_VP2/3_HSPG-KO. Another clone was picked, preped, test digested and sent for sequencing.

Freiburg10 pCerulean Zegfr1907 ML VP23HSPGKO.png


Conclusion: Sequencing results revealed positive results.

Picking clones of pGA14_MiddleLinker_VP2/3_insCap and pGA14_MiddleLinker_VP2/3_HSPG-KO

Investigator: Hanna

Unfortunately there grew a bacteria lawn over night - it was hardly not possible to pick clones. Nevertheless I tried and picked 2 clones of pGA14_MiddleLinker_VP2/3_insCap and pGA14_MiddleLinker_VP2/3_HSPG-KO.
To do: Mini-Prep and test digestion.

Sequencing results of pSB1C3_001_VP2/3_587-KO_BAP and pSB1C3_001_VP2/3_587-KO_6xHis

Investigator: Hanna

Comment: For the creation of our super constructs, the His-Tag and the BAP motif need to be cloned into VP2/3 for N-terminal fusion to VP2. Sequencing results showed that the 6xHis and BAP motif was not cloned into VP2/3_insCap.

Sample gallery
Freiburg10 Seq BAP his.png
Freiburg10 Seq BAP his2.png


To do: Clone 587-KO_6xHis and 587-KO_BAP into pSB1C3_001_VP2/3_insCap 1. via digestion of inserts and 2. via hybridization of referring oligos - digestion of vector with 800-900 ng.

Impressions of transfection of AAV293 with pCerulean_VP1up_NLS_mVenus_VP2/3

Investigator: Adrian

Yesterday AAV293 cells were transfected with pCerulean_VP1up_NLS_mVenus_VP2/3 and GMK-TK30 as gene of interest. Today, nuclear localization of the produced mVenus_VP2/3 fusion protein was visible and can be nicely seen in the following pictures:

Freiburg10 VP mVenus fusion localisation 5.jpg
Freiburg10 VP mVenus fusion localisation 7.jpg

















Conclusion: The VP2/3 fusion particle was transcribed and translated AND was transported back into the nucleus in order to be packaged by the ITR-flanked gene of interest.

Cloning of hGH_rITR into pSB1C3_lITR_phTERT_beta-globin_CFP and lITR_phTERT_beta-globin_mGMK_TK30 into pSB1C3_hGH_rITR

Investigator: Stefan

Vector name:

  • pSB1C3_lITR_phTERT_beta-globin_CFP (P682)
  • pSB1C3_hGH_rITR (P186)

Insert name:

  • pSB1C3_hGH_rITR (P186)
  • pSB1C3_lITR_phTERT_beta-globin_mGMK_TK30 (P672)
  • pSB1C3_lITR_phTERT_beta-globin_mGMK_TK30 (P673)


components volume for P186 /µlvolume of P186 X+E /µl volume of P672 + P673 /µl volume of P682 /µl
DNA 12 125 14
BSA (10x) 2,5 2 2 2
Buffer 4 (10x)3 2 2 2
Enzyme XbaI11 --
Enzyme PstI1- -1
Enzyme EcoI-1 1-
Enzyme SpeI-- 11
H2O669-
Total volume (e.g. 15,20,25,30 µl) 25 252020


Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt



Freiburg10 digestion hGH rITR.jpg



Comment: Since the first gel revealed problems with samples 672, 673 and 682 another digestion was prepared (same approach as shown above), this time using a 0,8% gel and another loading dye (without SDS).


0,4 g Agarose, 50 ml TAE (0,8%), 3 µl GELRED , at 115 Volt

Freiburg10 digestion hGH rITR backbones.jpg

Comment: Since sample 672 showed no bands this sample was discarded and cloning continued only using 673.




Gel extraction:
Was performed according to protocol.


T4 Ligation:
Ligation was performed over night.

ligation name 186 (X+E) + 673186 + 682
volume of vector 3,51 4,49
volume of insert5,38 2,64


Transformation:
Will be done tomorrow using BL21 cells.

New BL21 cells ready to use!

Investigator: Stefan

137. labday 02.10.2010

Repetition of the approach of subcloning the ViralBricks HSPG-ko BAP and His into VP2/3 with different strategies

Investigator: Bea

Comment: Unfortunately, the last experiment revealed that subcloning of the two ViralBricks did not work out which was shown by the sequencing results. Therefore we need to repeat the approach of subcloning the ViralBricks into the pSB1C3_VP2/3 which than can be used for fusing it to the n-terminal targeting approaches. Two different strategies will be carried out which means that we are digesting the pSB1C3_Viralbricks AND hybridize the oligos (BAP and His) in order to obtain some positive results.


The vector and ViralBricks were digested with BamHi and PvuII. The vector was dephosphorylated following the standard protocol. The vector was loaded on a 1% agarose gel before dephosphorylation was conducted. The results can be seen above in the gel picture:


Result:

Freiburg10 Digestion of VP23 inscap 02.10.2010.png

BioBrick Assembly of CD and mCherry

Investigator: Bea

Comment: We decided to choose some additional GOI´s for the vectorplasmid so the "new" GOI´s need to be fused to the leftITR_Promoter_betaglobin constructs. Since only one clone grew on the plate prepared by Kira where she fused the CD (cytosine deaminase) to the above mentioned construct I am going to repeat it aswell.


Digestion of the constructs:

  • P689 = pSB1C3_CD
  • P626 = iGEM_mCherry
  • P434 = pSB1C3_leftITR_CMV_betaglobin
  • P434 = pSB1C3_leftITR_phTERT_betaglobin
  • P525 = pSB1C3_VP2/capins


Freiburg10 Digestion of several constructs 02.10.2010.png


Loading plan: 

P689 (pSB1C3_CD)     P626 (iGEM mCherry)     P434 (pSB1C3_lITR_CMV_ß-globin)     P437 (pSB1C3_lITR_phTERT_ß-globin)


Results:

Freiburg10 Digestion of GOIs of vectorplasmid 02.10.2010.png


After gel extraction has been performed, the ligation was carried out.

Ligation:

Freiburg10 ligation GOI 02.10.2010.tif


The ligation mix was transformed into XL1-Blue cells and plated on agar plates containing chloramphenicol.

Next steps:
Picking clones and perform Mini-Prep. After the Mini-Preps have been performed this construct needs to be sequenced and tested in cell culture.


comment: just one clone? uhhhh, when did you take out the plate? i put it at around 1 am..maybe they need a while to grow, no? (Kira)
Yep, I found another clone ;-) But the two look pretty good. I took them out today in the afternoon - I guess that is enough time! But if you want I can put them back into the 37° C room??! (Bea)

Mini Prep and test digestion of pGa14_MiddleLinker_VP2/3_HSPG-KO

Investigator: Hanna
Comment: In order to fuse the DARPin to the N-terminus of VP2/3_insCap and VP2/3_HSPG-KO, these motifs need to be fused to the Middle Linker (performed on 30.9.2010). In order to be able to immediately continue with cloning, BL21 cells were transformed and clones were picked in the noon. Now, the DNA needs to be preped and test digested in order to perform a 3-fragment-ligation with the DARPin and the pSB1C3_001_CMV plasmid. Unfortunately the cells of the VP2/3_insCap construct grew not densely enough. Therefore two new clones were picked from the plate and were inoculated in 10 mL LB medium. The other construct (pGa14_MiddleLinker_VP2/3_HSPG-KO) was preped and test digested.

TEST DIGESTION: For both clones:

  • DNA: 4 µL
  • Buffer 4: 1 µL
  • BSA: 1 µL
  • EcoRI: 0.5 µL
  • PstI: 0.5 µL
  • H2O: 3 µL


Incubation: 1 hour.
Agarose gel: 0.8 %, 115 Volt, 1 hour.
Comment: The choice of these restriction enzymes was not clever, because if cloning didn't work, I expect one band at 2379 bp (the insert should hardly be seen) - if it worked one band at 2379 bp and a second at 2005 bp. Fortunately the bands could be separated ans revealed positive results for both clones :
Freiburg10 2 10 TestD.png

Next steps:

  • Mini-Prep and test digestion of pGa14_MiddleLinker_VP2/3_ins Cap.
  • 3 fragment ligation of MiddleLinker_VP2/3_ins Cap or MiddleLinker_VP2/3_HSPG-KO + DARPin + pSB1C3_001_CMV backbone.

Sequencing analysis of HSPG-ko in several IRCK constructs

Investigator: Stefan

pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10 clone 1 (P687):
Freiburg10 sequencing results P687.JPG

pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10 clone 2(P688):
Freiburg10 sequencing results P688.JPG


pSB1C3_001_RC_IRCK_VP1-ko_HSPG-ko_P5tataless cl1 (P644):
Freiburg10 sequencing results P644.JPG


pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1 (P646):
Freiburg10 sequencing results P646.JPG


Comment: Sequencing results show that all plasmids contain the HSGP-ko. Working will be continued using P644, P646 and P687.


Mini Prep of pGa14_MiddleLinker_VP2/3_inscap and pSB1C3_Darpin

Investigator: Jessica

  • pSB1C3_Darpin clone1 c=158.5 ng/µl
  • pSB1C3_Darpin clone2 c=205.6 ng/µl
  • pGA14_MiddleLinker_VP2/3_inscap clone 1 c=147.5 ng/µl
  • pGA14_MiddleLinker_VP2/3_inscap clone 2 c=121.0 ng/µl

Test-digestion:

Components pSB1C3_Darpin clone1/2
DNA 1,5 µl
BSA (10x) 1 µl
Buffer no. 4 1 µl
Enzyme 1 XbaI 0,4 µl
Enzyme 2 AgeI 0,4 µl
H2O 5,7 µl
Total volume 10


Hanna wrote that a 3rd enzyme is need for the test digestion of pGA14_MiddleLinker_VP2/3_inscap but I couln't find any sequence of pGA14_MiddleLinker_VP2/3_inscap or pGA14

You can either find these constructs in my workingfolder "DARPin" or the pGA14_MiddleLinker in the "Linker-Folder" and the VP2/3_insCap in many workingfolders!!!! You could have also digest the construct with EcoRI and SpeI... then you would expect a 2 and a 2.3 kb fragment... menas: you have to run the gel at least 1 hour - but it works (see the previous test digestion of the MiddleLinker_VP2/3_insCap construct!!!!).

Freiburg10 testdigestdarpin.jpg


Test digestion of pGA14_MiddleLinker_VP2/3_inscap

Investigator: Achim

  • pGA14_MiddleLinker_VP2/3_inscap clone 1 c=147.5 ng/µl (P698)
  • pGA14_MiddleLinker_VP2/3_inscap clone 2 c=121.0 ng/µl (P699)
  • Control: pGA14_MiddleLinker (P301)
  • Digestion with EcoRI & SalI; should yield two bands of 3400 & 1000 bp. The negative control should only be cut once.
Both samples show correctly sized bands. The negative control ran further than expected, it should be located at 4500 bp.

Mass Midi-Prep III

Investigator: Chris W.

Midi-Prep of:


pSB1C3_001_RC_IRCK_VP1-ko_HSPG-ko_P5tataless cl1 =P700 =B521
pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1 =P701 =B523
pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10 clone 1 =P702 =B561
pCerulean_ZEGFR:1907_MiddleLinker_VP2/3_HSPG-KO clone 2 =P703 =B543
pCerulean_CFP_MiddleLinker_VP2/3_HSPG-KO =P704 =B507
pCerulean_ZEGFR:1907_SEG_VP2/3_HSPG-KO =P705 =B509
pCerulean_ZEGFR:1907_ShortLinker_VP2/3_HSPG-KO =P706 =B510
pCerulean_ZEGFR:1907_LongLinker_VP2/3_HSPG-KO =P707 =B511
pCerulean_6xHis_MiddleLinker_VP2/3_HSPG-KO =P708 =B512
pCerulean_VP1up_NLS_ZEGFR:1907_VP2/3_HSPG-KO =P709 =B513
pCerulean_VP1up_NLS_6xHis_VP2/3_HSPG-KO =P710 =B514
pCerulean_VP1up_NLS_mVenus_VP2/3_HSPG-KO =P711 =B515
pSB1C3_001_RC_IRCK_P5tataless clone 1 =P712 =B516


The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no. P700P701P702P703P704P705P706P707P708P709P710P711P712
concentration (ng/µl)695 370 6011548399636302460261018602913296430301200



Analysis of viral harvest via spectrometer

Investigator: Adrian, Patrick
The motivation: We want to figure out if it is possible to purify our viral stocks via centrifugation with 10.000 g and/or 20.000 g. In theory/ according to the literature the viral particles should be in the cells and attached to the HSPGs at the cellular surfaces.
The plan: Two different viral stocks were prepared:

Stock 1 with VP2-mVenus-fusion-Capsid loaded with TKGMK (4 wells from a 6 well plate)


Stock 2 "standard" virus (4 wells from a 6 well plate)

These two stocks got harvested according to standard protocol (scratching cells, transfer them in to 15 ml falcons, resuling 12ml solution total). After centrifugation (10 min 200g), the supernatant got transferred into two new 15 ml falcons and the pellets were resuspended with 10 ml DMEM Medium. The Stocks got freezed thawed two times so finally there were:

  1. DMEM as negative control
  2. resuspended Pellet with mVenus-fusioned viral capsids
  3. resuspended Pellet with "standard virus"
  4. supernatant with mVenus-fusioned viral capsids
  5. supernatant with "standard virus"


The fluorescence of each stock was measured via spectrometer. After that, stocks 2 and 4 were centrifugated 10min with 10.000 g followed by an other fluorescence measuring. Finally the stocks got spin down with 20.000 g for 10 min
The results:
Insert fluorescence spectrum

The conclusions: The highest amount of fluorescence was measured in the supernatant, the individual centrifugation steps had no decrease in fluorescence as consequence!


Keep in mind, without further investigation it is not valid to say that we can purify our stocks via 10-20.000 g cantrifugation steps, because we dont know if the viral capsids are still intact! The FACS analysis are not sufficient enough to make a decision, because our last samples had YFP as GOI (=> so efficient cell sorting was not possible). We need to do qPCRs to make a valid evidence.

200µl-Transduction for testing the pTERT-promoter

Investigator: Adrian, Patrick
We transduced A431, HT1080 and AAV293 cells with 4 different viral stocks:

  1. control vector from 15.9, 750.000 cells, 3.3 µg approach
  2. pTERT_mVEnus-XXXX
  3. pTERT_mVEnus-XXXX
  4. His-linker-fusion

CRUCIAL: There was not enough viral stock: so the whole Transduction was performed with 200µl instead of 1000µl

There are 45 samples to FACS on monday 4.10. Tripple values for each stock of each cell lines.

Transfection

Investigators: Adrian, Patrick
Transfection followed standard protocol (3.3 µg of each plasmid, 1 ml 0.3M CaCl2 and 1 ml of 2xHBS buffer PH 11.12) but with 20 min incubation time (Ca-DNA-2xHBS)
following stocks:

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