Team:Freiburg Bioware/NoteBook/Labjournal/October

From 2010.igem.org

136. labday 01.10.2010

Test Digestions of yesterdays minipreps

Investigator: Achim, Anna

• 001_CMV: Cut with EcoRI, PstI & PvuII
  • P654 (Lane 1)
  • P655 (Lane 2)
  • P686 (Lane 3)
  • Control P320 (Lane 4)

• CMV_affi_VP2/3_capins: Cut with EcoRI & SspI
  • P678 (Lane 5)
  • P679 (Lane 6)
  • Control P514 (Lane 7)
    • Results:The expected bands should run at 1700 and 3000 bp. P679 was sent for sequencing.

• CMV_affi_VP2/3_capins_HSPG-KO: Cut with EcoRI & SspI
  • P680 (Lane 8)
  • P681 (Lane 9)
  • P684 (Lane 10)
  • P685 (Lane 11)
  • Control P613 (Lane 12)
    • Results:The expected bands should run at 1700 and 3000 bp. P684 was sent for sequencing.

• 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)

I guess that the pSB1C3_001_CMV did NOT work since the control with BLA looks nearly the same. We should repeat the experiment by cloning the CMV promoter into another vector. I would recommend it because we have a lot of constructs which we should fuse to the CMV promoter and in order to avoid the three-fragment ligation.

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

incubation @ 37 C for approx. 2 h

1% agarose gel

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

incubation @ 37 C for approx. 2 h

1% agarose gel

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.

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

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:

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)

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

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

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.

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:

The ligation was performed over night at 18°C with T4-ligase.

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

Loading plan:

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

Results:

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

Ligation:

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 :


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):

pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10 clone 2(P688):

pSB1C3_001_RC_IRCK_VP1-ko_HSPG-ko_P5tataless cl1 (P644):

pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1 (P646):

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:

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!!!!).

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: