Team:Freiburg Bioware/NoteBook/Labjournal/September

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September

107. labday 01.09.2010

Loop insertion BioBricks: Sequencing results

Investigator: Achim, Anna

Comment: Sequencing results of Loop insertion BioBricks from 31.08.10; the following clones were sent for sequencing:


  • 2: 453 BAP: No Insert

Freiburg10 453 bap.JPG

  • 3:453 His: Insert okay

Freiburg10 453 his.JPG

  • 5: 453 RGD: Mutation in 453 upstream region

Freiburg10 453 rgd.JPG

  • 7: 587 BAP: Mutation in Bap insert

Freiburg10 587 bap.JPG

  • 9:587 His: Insert okay

Freiburg10 587 his.JPG

  • 11:587 KO BAP : Insert okay

Freiburg10 587 KO bap.JPG

  • 13:587 KO empty : Insert okay

Freiburg10 587 KO empty.JPG

  • 16: 587 KO his: Mutation in 587 KO

Freiburg10 587 ko his.JPG

  • 17: 587 KO rgd: No insert

Freiburg10 587 KO rgd.JPG

  • 19 :587 RGD: Insert okay

Freiburg10 587 rgd.JPG

  • 25: 587 Z34C: Mutation in downstream 587 region

Freiburg10 587 z34c.JPG

Mini-Preps:

New Mini-Preps of the samples with no/mutated insert were prepared. The following clones were used:

Components 453 BAP 587 BAP 453 RGD 587 KO RGD 587 KO HIS 453 Z34C 587 Z34C 587 KO Z34C 587 KO Z34C SPACER
Clone1.3 + 1.4 2.3 + 2.4 4.3 + 4.4 6.3 + 6.4 9.3 + 9.4 11.3 + 11.412.2 + 12.4 13.2 + 13.4 14.3 + 14.4


Comment: Samples 1.3, 2.3, 4.3, 6.3, 9.3, 11.3, 12.2, 13.2, 14.3 were sent for sequencing. For sequencing results go to labday 02.09.10.

.

Design of primers for pAAV_RC

Investigator: Bea

Primers for different cloning strategies of the synthesized cap gene into pAAV_Rc have been designed.

  1. The first primers are mutagenesis primers which delete the KpnI recognition site at position 3968. These primers can (or should be used) be used if cloning with BspMI/BfuI (isochizomer) does not cut the pAAV_RC efficiently. Instead of using this enzyme we can use XcmI to subclone the cap gene with performing a site-directed mutagenesis afterwards with designed primers.
  2. The second idea is to design oligoduplexes (hybridized oligos) which contain the recognition site for BspMI. In Gormley et al (2002) the idea was to add oligodupexes with the recognition site for BspMI and therefore provide the "second recognition site" in trans.

Next step: Check primers and order them today! Designed primers are stored in my Workingfolder under paav_rc and a word document was created whcih can be found under Oligos --> primers for different strategies...

Sequencing results of pSB1C3_Zegfr:1907_Linker

Investigator: Hanna

Comment: Sequencing results looked all well. One bp was missing in the theoretical sequence of SEG suffix - but is correct in the actual sequence.


pSB1Cr_Zegfr:1907_LongLinker:
PSB1C3 Zegfr1907 LongLinker.JPG

pSB1Cr_Zegfr:1907_SEG:
PSB1C3 Zegfr1907 SEG.JPG

pSB1Cr_Zegfr:1907_MiddleLinker:
PSB1C3 Zegfr1907 MiddleLinker.JPG

pSB1Cr_Zegfr:1907_ShortLinker:
PSB1C3 Zegfr1907 ShortLinker.JPG

These constructs will be cloned into pCerulean for N-terminal fusion to VP2.
go to the top ;)

Trafo evalutaion and Inoculation

Investigator: Kira
The plate with transformed CD-biobrick contains around 40 colonies. 4 of them will be inoculated into LB and incubated @37 C over-night.
go to the top ;)

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

Investigator: Patrick


The two Ligations were performed according to the standard protocol:
1 µl T4 DNA ligase, 1 µl 10x Buffer, 4,6 µl vector (P273), 3,34 µl Insert (P276, upper and lower band).
Incubation time: 50 minutes.

During the transformation i forgot to put the cells into the 37°C shaker so i incubated them afterwards again for 45 minutes at 37 °C on a shaker.

The mutual conles will be picked tomorrow to inoculate 10 ml DYT following a mini-prep on 03.09.2010

Transfection, Seeding AAV293 and HT1080

Investigator: Kerstin

  • Transfection: 10 plates, same treatment (10µg of RC (P357), pHelper (P356)and mVenus (P263), following the standard protocol)
  • Seeding HT1080 for transduction with virus from 21.08.2010 (10µg of RC, pHelper, TKGMK; pH=7,10)
  • Seeding AAV293 for transfection with plasmids without HgH or beta-globin (verifying functionality)

Cloning of ZEGFR:1907_Middle-Linker,ZEGFR:1907_Short-Linker, ZEGFR:1907_SEG-Linker and ZEGFR:1907_Long-Linker into pCerulean

Investigator: Stefan

Comment:


Digestion:

components Vector (P273) ZEGFR:1907_Middle-Linker (P290) ZEGFR:1907_Short-Linker (P292) ZEGFR:1907_SEG-Linker (P296) ZEGFR:1907_Long-Linker (P298)
DNA 3,8 8,8 10,2 9,2 8,7
BSA (10x) 2 2 2 2 2
Buffer 4 (10x)2 2 2 2 2
XbaI 1 1111
PstI 11111
H2O10,2 5,2 3,8 4,8 5,3
Total volume 20 20 20 20 20


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



Freiburg10 pCerulean affi linker.jpg




Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

  • P273: c= 3,1 ng/µl
  • P290: c= 3,2 ng/µl
  • P292: c= 1,8 ng/µl
  • P296: c= 3,0 ng/µl
  • P298: c= 3,2 ng/µl


T4 Ligation:

The Ligation was performed as following:

Comment: No sufficent vector concentration, therefore 5 µl of vector was used for every approach.

  • Vector Volume: 5 µl
  • Insert Volume: 3 µl


  • 1µl T4-Ligase buffer (10x)
  • 8µl (Vector + Insert) mix
  • 1µl T4-Ligase


Incubating for 40 minutes.


Transformation:

Trafo was performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol


108. labday 02.09.2010

Sequencing of loop insertions BioBricks: 2nd round

Investigator: Achim, Anna

We analyzed the new sequencing results and found two correct sequences:

  • 587 BAP clone 2.3
  • 453 RGD clone 4.3

The other samples didn't contain any inserts or had wrong insertions. Apparently, the vector tends to religate easily. A possible reason for the religation of the incompatible ends is the overnight ligation at 18°C. We sent preps of the remaining 7 ligations for sequencing and will prep new clones or repeat the ligation tomorrow, depending on the new sequences.

Comment: Clones 1.4, 6.4, 9.4, 11.4, 12.4, 13.4, 14.4 were sent for sequencing. For sequencing results go to labday 03.09.10.

.

Mini-preps of pSB1C3_SV40 and pCerulean_VP1_NLS

Investigator: Bea (Chris L)

Mini-Prep was performed according to the standard protocol

  • P363 = pSB1C3_SV40 clone 1 = 277,70 ng/µl
  • P364 = pSB1C3_SV40 clone 2 = 265,01 ng/µl
  • P365 = pCerulean_VP1up_NLS (1) clone 1 = 473,14 ng/µl
  • P366 = pCerulean_VP1up_NLS (1) clone 2 = 455,88 ng/µl
  • P367 = pCerulean_VP1up_NLS (1) clone 3 = 482,86 ng/µl
  • P368 = pCerulean_VP1up_NLS (2) clone 1 = 438,52 ng/µl
  • P369 = pCerulean_VP1up_NLS (2) clone 2 = 478,00 ng/µl
  • P370 = pCerulean_VP1up_NLS (2) clone 3 = 419,00 ng/µl
    Construct were digested with XbaI and PstI for 45 minutes at 37°C. The loading plan on the 1% agarose gel looks like this:

    _M_ ___ _363_ ___ _364_ _365_ _366_ _367_ _368_ _369_ _370_ ___

    Results: P365 was sent for sequencing because test digestion looks goos. In contrast to the SV40 appraoch. This needs to be repeated tomorrow.
    • Plasmid: pCerulean_VP1up_NLS
    • Plasmid number: P365
    • Tube name: SB1
    • Primer used: CMV-F

    Mini-Prep and test digestion of pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker

    Investigator: Hanna

    Comment: Zegfr:1907 (Affibody) and the His-Tag which was coupled to the middle linker were cloned into pCerulean. 3 clones of each construct were picked yesterday.


    Plasmid Mini-Prep

    • new vector name: pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker


    Glycerol Stocks
    1. pCerulean_Zegfr:1907

    Clone 1 Clone 2 Clone 3
    Bacteria strain XL1b XL1b XL1b
    Plasmidname pCerulean_Zegfr:1907 pCerulean_Zegfr:1907 pCerulean_Zegfr:1907
    Date 2.9.10 2.9.10 2.9.10
    given glycerol-stock no. B275 B276 B277
    given plasmid no. P371 P372 P373


    2. pCerulean_6xHis_MiddleLinker

    Clone 1 Clone 2 Clone 3
    Bacteria strain XL1b XL1b XL1b
    Plasmidname pCerulean_6xHis_MiddleLinker pCerulean_6xHis_MiddleLinker pCerulean_6xHis_MiddleLinker
    Date 2.9.10 2.9.10 2.9.10
    given glycerol-stock no. B278 B279 B280
    given plasmid no. P374 P375 P376


    Test digestion

    • buffer used: 4; Restriction-enzymes used: Enzyme 1 PstI-HF ; Enzyme 2 EcoRI-HF
    • Plasmid
      • Given Plasmid-Number: 371; DNA concentration: 381.3 ng/µL;
      • Given Plasmid-Number: 372; DNA concentration: 377.5 ng/µL;
      • Given Plasmid-Number: 373; DNA concentration: 409.7 ng/µL;
      • Given Plasmid-Number: 374; DNA concentration: 404.2 ng/µL;
      • Given Plasmid-Number: 375; DNA concentration: 366.1 ng/µL;
      • Given Plasmid-Number: 376; DNA concentration: 375.0 ng/µL;


    Comments::)

    Pipetting scheme for each test digestion:

    Components Volume/µL
    DNA 2.7
    BSA (10x) 1
    Buffer no. 4 (10x) 1
    EcoRI-HF 0.5
    PstI-HF 0.5
    H2O 4.3
    Total volume 10


    • Incubation: 55 minutes


    Agarose-Gel:


    0.875 g Agarose, 50 mL TAE (1.75 %), 3 µL EthBr, at 115 Volt, running time: 35 minutes

    Sample Sample/µl] Loading dye (6x)/µl Expected size
    pCerulean_Zegfr:1907 clone 1 10 µl 2 µl 231 bp
    pCerulean_Zegfr:1907 clone 2 10 µl 2 µl 231 bp
    pCerulean_Zegfr:1907 clone 3 10 µl 2 µl 231 bp
    pCerulean_6xHis_MiddleLinker clone 1 10 µl 2 µl 105 bp
    pCerulean_6xHis_MiddleLinker clone 2 10 µl 2 µl 105 bp
    pCerulean_6xHis_MiddleLinker clone 3 10 µl 2 µl 105 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample P371 /µl Sample P372 /µl Sample P373 /µl Sample P374 /µl Sample P375 /µl Sample P376 /µl
    Lane 5 12 12 12 12 12 12


    Comments: Test digestion looked well:

    Freiburg10 pCerulean Zegfr1907andpCerulean 6xHis ML.png
    Clone 1 of each construct were sent for sequencing (P371 and P374 = HW1 and HW2). Used primer: GATC_std_CMV-F.

    Midi-Prep of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1 & pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1

    Investigators: Chris W.

    Midi-Preps of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1=P377 and pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1=P378

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

    plasmid-no. P377P378
    concentration (ng/µl)325,04 561,15


    Mini-Prep and test digestion of the CD biobricks

    Investigator: Kira

    Mistake.png
    Ohne Worte *lach*

    Wh did you digest with eco and ndeI?? is there a spechía reasonn for it?? if the coonstruct was in the rfc standard could digest it with the igem standard enzymes??

    components sample /µl
    DNA 2,5
    BSA (100x) 0
    Buffer ___4_ (10x) 1,5
    Enzyme NdeI (no.Lab:___) 0,5
    Enzyme EcoRI (no.Lab:___)0,5
    H2O10
    Total volume (e.g. 15,20,25,30 µl) |15
  • Incubation: 1 h

    Freiburg10 test digestion of CD.jpg

    Sample 1 was sent for sequencing

    109. labday 03.09.2010

    Miniprep and test digestion of pSB1C3_lITR_pTERT_ßglobin_mVenus_hGH_rITR

    Investigator: Achim

    Comment: The test digestion did not show the expected fragments of 2000 and 2500 bp. the photo was exposed too long to see distinct bands. Digestion will be repeated.

    AA1.png

    Sequencing results

    Investigator: Hanna

    Comment: pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker were cloned and sent for sequencing yesterday.

    1. pCerulean_Zegfr:1907: Sequencing looked well.
    Freiburg10 pCerulean Zegfr1907 seq.JPG

    2. pCerulean_6xHis_MiddleLinker: Sequencing looked also well. Freiburg10 pCeruelan 6xHis MiddleLinker seq.JPG

    Cloning of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

    Investigator: Hanna

    Comment: For N-terminal fusion to VP2, the Affibody - fused to different kinds of linkers - will be cloned into the expression plasmid pCerulean. For imaging CFP, which was fused to the middle linker, will be cloned into pCerulean.



    Practical Cloning:

    • plasmid:
      • Vector: name: pCerulean number: P273
      • Insert: name: pSB1C3_Zegfr:1907_LongLinker (P298), pSB1C3_Zegfr:1907_MiddleLinker (P290), pSB1C3_Zegfr:1907_SEG (P296), pSB1C3_Zegfr:1907_ShortLinker (P292), pSB1C3_CFP_MiddleLinker (P276)
    • new vector name: pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker
    • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 EcoRI-HF ; Enzyme 2 PstI-HF
    • DNA concentration (vector): 419.5 ng/µL ; DNA concentration (insert): P298 229.7 ng/µL, P290 227.4 ng/µl; P296 218.7 ng/µL; P292 196.6 ng/µL; P276 207.7 ng/µL


    Comments: No.

    Digestion

    components volume of pCerulean /µl volume of Zegfr:1907_MiddleLinker /µl volume of CFP_MiddleLinker /µL
    DNA 8.3 7.6 4.8
    BSA (10x) - - -
    Buffer 4 (10x) 2 2 2
    Enzyme 1 EcoRI-HF 1 1 1
    Enzyme 2 PstI-HF 1 1 1
    H2O 7.7 8.4 11.2
    Total volume 20 20 20
    • Incubation: 1.5 h



    Agarose-Gel:


    0.8 g Agarose, 50 TAE (1.5 %), 5 µL EthBr, at 70 Volt, running time: ~ 1 hour

    Sample Sample/µl] Loading dye (6x)/µl Expected size 1
    P273 20 µl 4 µl 3922 bp
    P298 20 µl 4 µl 273 bp
    P290 20 µl 4 µl 261 bp
    P296 20 µl 4 µl 345 bp
    P292 20 µl 4 µl 249 bp
    P276 20 µl 4 µl 801 bp


    • Marker: GeneRuler ladder mix
    Marker Sample P273 /µl Sample P276 /µl Sample P290 /µl Sample P292 /µl Sample P296 /µl Sample P298 /µl
    Lane 5 24 24 24 24 24 24



    Gel extraction


    Gel measurement:

    Sample Volume Concentration
    P273 20 115.45 ng/µL
    P276 20 9.91 ng/µL
    P298 20 5.07 ng/µL
    P296 20 9.03 ng/µL
    P290 20 10.31 ng/µL
    P292 20 20.78 ng/µL




    Ligation


    1. PCerulean_Zegfr:1907_LongLinker

    P298 P273
    Volume/µl 6.61 1.39


    2. PCerulean_Zegfr:1907_MiddleLinker

    P290 P273
    Volume/µl 5.53 2.47


    3. PCerulean_Zegfr:1907_SEG

    P296 P273
    Volume/µl 6.17 1.83


    4. PCerulean_Zegfr:1907_ShortLinker

    P292 P273
    Volume/µl 4.11 3.89


    5. PCerulean_CFP_MiddleLinker

    P276 P273
    Volume/µl 7.02 0.98


    Trafo


    Trafo was performed following the standard protocol. Used cells: XL1b, DNA amount: 2.5 µL of each ligation reaction. Plates were stored @ 37°C room over night.

    Harvest viral particles, Seeding HT1080 cells

    Investigator: Adrian, Kerstin

    • Harvest viral particles of Transfection from 31.08.2010 (six approaches: 10µg of RC (2x P326 and 2x P325 and 2x Stratagene), pHelper and GOI (P262))
    • Seeding HT1080 cells

    Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

    Investigator: Patrick


    The Miniprep was performed according to the standard protocol. Labelling:

    • P381: pCerulean_middlelinker clone 1 upper band : 526,0 ng/µl
    • P382: pCerulean_middlelinker clone 2 upper band : 522,0 ng/µl
    • P383: pCerulean_middlelinker clone 3 upper band : 463,0 ng/µl
    • P384: pCerulean_middlelinker clone 1 lower band : 465,0 ng/µl
    • P385: pCerulean_middlelinker clone 2 lower band : 500,0 ng/µl
    • P386: pCerulean_middlelinker clone 3 lower band : 524,0 ng/µl
    Öhm Patrick: Hier ist der Grund, warum das Experiment "wiederholt" wurde: Falsche Beschrieftung hier, als auch in der Excel Tabelle ;) "Finde den Fehler!" :P


    see also http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/August_2010#Cloning_CFP_middlelinker_.28from_pSB1C3_CFP_middlelinker.2C_P276.29_into_pCerulean_.28P273.29

    ;-) bitte ausfüllen

    Testdigestion: 7 µl DNA sample, 1 µl Buffer 4 (10x), 1 µl BSA, 0,5 µl Xba, 0,5 µl PstI-HF
    Expected size of the fragments: 786 & 2053bp
    There seems to be something wrong.

    Freiburg10 pat20100903.jpg

    Sent for sequencing: P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev
    Used primer: O21 : CMV_reverse_qPCR

    Cloning of pSB1C3_leftITR_pTert and pSB1C3_mGMK with pSB1C3_leftITR_CMV

    Investigator: Chris L.

    • Vector: name: pSB1C3_leftITR_pTERT clone 1 P256 c=179,2 ng/µl
    • Vector: name: pSB1C3_leftITR_CMV P188 c=231,6 ng/µl
    • Insert: name: pSB1C3_mGMK P195 c=258,6 ng/µl
    • new vector name: pSB1C3_leftITR_CMV_GMK
    • new vector name: pSB1C3_leftITR_pTERT_GMK
    • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 PstI ; Enzyme 2 SpeI ; Enzyme 3 XbaI


    components P195 P256 /µl P188 /µl
    DNA 7,7 11,2 8,6
    BSA (10x) 2 2 2
    Buffer 4 (10x)2 2 2
    Enzyme SpeI 1 1 0
    Enzyme XbaI 0 0 1
    Enzyme PstI 1 1 1
    H2O 6,3 2,8 5,4
    Total volume (e.g. 15,20,25,30 µl) 2020 20


    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 130 Volt, running time:45

    Results:
    Freiburg10 pSB1C3 lITR pTERT and pSB1C3 lITR CMV with GMK Insert.jpg

    • c(Insert)= 15,37 ng/µl; size: 627 bp
    • c(Vector)= 50,84 ng/µl; size: 2869 bp
    • c(Vector)= 75,64 ng/µl; size: 2672 bp


    • Ligation of PCR products and vector:

    For the Ligation 1µl T4 buffer (10x) and 1µl T4 ligase were used. Incubation time: 45 min

    pSB1C3_leftITR_CMV + GMK /µl pSB1C3_leftITR_pTERT + GMK
    Vector 5,48 6,21
    Insert 2,52 1,79


    • Transformation:

    The transformation was done following the standard protocol using XL1 blue cells.

    Sequence analysis of pCerulean_VP1up

    Investigator: Bea

    The assembled pCerulean_VP1up which serves as the first construct in the VP1 insertion cloning assembly was sent for sequencing.

    • Plasmid used: P365
    • Primer used: CMV-F
    • Tube name: SB1
    • Folder (Geneious): N-terminal Targeting --> pCerulean_VP1up_NLS



    pCerulean_VP1up_NLS_Targeting molecule

    Investigator: Bea

    Comment: For preparing the next step in the VP1 insertion, different targeting molecules will be fused to the construct pCerulean_VP1up_NLS.

    1. The first construct is the Affibody ZEGF-R:1907 which was already cloned into the standard iGEM plasmid and can be sued for target tumor cells overexpressing the EGF receptor.
    2. The second construct is the mVenus (Yellow fluorescent protein) which can be used for imaging experiments
    3. Another motif will be the His-tag. Since the His-tag is very small, the oligos will be hybridized and directly ligated into the pCerulean backbone.


    Digestion of vector:

    • Plasmid used: pCerulean_VP1up_NLS (P365) c=470 ng/µL
    • Add BSA
    • AgeI-HF and SpeI-HF were used


    Protocol of the digestion of the vector:

    Components vpCerulean_VP1up_NLS/µL
    DNA 3
    BSA (100x) 2,5
    Buffer no. 4 (10x) 2,5
    Enzyme 1 AgeI 1
    Enzyme 2 SpeI HF 1
    H2O 15
    Total volume 25


    • Incubation of the sample at 37°C
    • Incubation for 120 minutes


    The Targeting molecules were digested with different enzmyes than the vector for providing the ability to assemble the different constructs together without creating new restriction site but to delete them. This works by fusing NgoMIv and AgeI together.

    Digestion of the targeting molecules

    • Plasmids used:
      1. pSB1C3_zEGF-R:1907 (P284) c=146 ng/µL
      2. pGA14_mVenus(P60) c=280 ng/µL
    • Add BSA
    • NgoMIV and SpeI-HF were used


    Components vZEGFR (P284)/µL vpGA_mVenus (P60)/µL
    DNA 13 µl 7 µl
    BSA (10x) 2,5 µl 2,5 µl
    Buffer no. 4 (10x) 2,5 µl 2,5 µl
    Enzyme 1 NgoMIV- 1,0 µl 1,0 µl
    Enzyme 2 AgeI HF 1,0 µl 1,0 µl
    H2O 15 µl 15 µl
    Total volume 25 25
    • Incubation of the sample at 37°C
    • Incubation for 90 minutes


    After incubation the samples were loaded on a 1% agarose gel. The obtained gel (after running the gel for 15 minutes) can be seen below.


    Results: The boxes mark fragments which ere cut out of the gel. The left lane (pCerulean) looks a bit strange, because Marker was added after the digestion reaction instead of Loading dye.

    Parallel, for assembling the His-Tag to the construct pCerulean_VP1up_NLS, oligos for the His-Tag in the RFC25 standard (provided by Gerrit) were hybridized and can directly be ligated in the digested vector. This strategy was used because of the smal size of the His-Tag which cannot be separated easily by the agarose gel.


    Hybridization of the His-Oligos

    • 10µL oligo1 (1:10)
    • 10µL oligo2 (1:10)
    • 4µL 100mM Tris-Cl pH 8
    • 10µL 5mM MgCl2
    • 8µL H2O

    The used programm for the hybridization:

    • 99°C 7´
    • 99°C 1´
    • -1°C R=0,3°/s
    • Goto 2 rep 74
    • Hold 4°C


    After the gel extraction of a T4 ligation was performed.

      Affibody ZEGF-R:1907:

      vCerulean_VP1up_NLS = 5,82µL
      vZEGF-R:1907 = 2,18µL

      mVenus Z:

      vCerulean_VP1up_NLS = 4,04µL
      vmVenus = 3,96 µL

      6xHisTag:

      vCerulean_VP1up_NLS = 0,2µL
      v6xHisTag = 7,98µL


    Next steps: Picking clones of the trafo plates (containing Kanamycin) and perform the Mini-Prep. The obtaining constructs finally will be fused to the VP2/3 protein which results in the final construct for the VP1 targeting approach.

    3rd Repetition of Mini-Preps and test digestion for Loop insertion BioBricks

    Investigator: Achim, Anna

    Sequencing results of Loop insertion BioBricks from 02.09.10; the following clones looked well:

    • pSB1C3_587_KO_RGD_clone6.4
    • PSB1C3_587_KO_His_clone9.4


    Update: 9 of the 14 ViralBricks looked well (sequencing results from 01. - 03.09.). Two new clones of the remaining constructs were prepared and test digested.

    Test digestion:

    components Volume for each sample /µl
    DNA 10
    BSA (10x) 1,5
    Buffer 4 (10x) 1,5
    Enzyme EcoRI HF 0,5
    Enzyme NotI HF 0,5
    H2O 1
    Total volume /µl15


    Components 453 BAP 453 Z34C 587 Z34C 587 KO Z34C 587 KO Z34C SPACER
    Clone1.5 + 1.6 11.5 + 11.6 12.5 + 12.6 13.5 + 13.6 14.5 + 14.6


    Incubation time: 1 h, Incubation temperature: 37°

    Preparation of gel:
    1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

    3.9.10 AA.png


    Expected fragment sizes /bp:
    pSB1C3: 2051 bp
    BAP: ~150
    Z34C: ~ 200

    Comment: The following clones were sent for sequencing: 1.6, 12.6 ,13.6 and 14.5.
    To do: Retrafo of clone6.4 and clone9.4 and preparation of glycerol stocks.

    Cloning of Cap into pAAV_RC_ins-rep: preparation for SDM

    Investigator: Stefan

    Comment: Cloning Cap into pAAV still does not work as planned. As another approach Cap can be cut using BsiWI and XcmI and performing a SDM in the part of Cap not cloned into pAAV. Primers are ordered and should arrive on monday to proceed.


    Digestion:

    components pMA_RepCap Vector_SDM_InsPvuII (P211) pAAV_RC_ins-rep (P250)
    DNA 7,9 2,1
    Buffer 2 (10x)2 2
    XcmI 1 1
    BsiWI 11
    H2O8,1 13,9
    Total volume 20 20


    Comment:Digestion was performed using two steps, first incubating for 1 hour at 37 °C, afterwards for 1,5 hours at 55 °C.



    Gel:

    0,5 g Agarose,50 ml TAE (1%), 5 µl EtBr , at 115 Volt, running time: 50 minutes



    Freiburg10 pCerulean pMA pAAV.png




    Gelextraction:

    The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

    • pMA_RepCap Vector_SDM_InsPvuII (P211): c= 6,26 ng/µl
    • pAAV_RC_ins-rep (P250): c= 13,72 ng/µl


    Quick Ligation:

    Comment: XcmI produces only 1 base overhang, therefore Quickligase was used for ligation and incubation time increased.

    The Ligation was performed as following:

    • Vector Volume: 4,59 µl
    • Insert Volume: 4,41 µl


    • 10 µl QuickLigase buffer (2x)
    • 9 µl (Vector + Insert) mix
    • 1 µl QuickLigase


    Incubating for 60 minutes.


    Transformation:

    Trafo was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with ampicilin.

    110. labday 04.09.2010

    Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273): Sequencing results of P381 and P385

    Investigator:Patrick

    Sequencing results of P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev: A wrong primer was used so there are no relevant sequence data yet. On sunday i will sent these clones again for sequencing. Primer: GATC_std_CMV-F

    Ha ich war schneller als Bea beim "Kopfkratzen-setzen" *lol*

    Hiermit verspreche ich dass es noch jede Menge Kopfkratzer, Laboraufsichtswichtel und pinke Panter geben wird (Patrick)









    Harvest viral particles, Transduction

    Investigator:Kerstin, Adrian

    • Harvest viral paricles from one plate of transfection from 01.09.2010 (10 plates, same treatment (10µg of each plasmid P357, P263, P356))
    • Transduction:
    1. 1x 6-well: 0,5ml of virus from 01.09.2010 (testing R/C P326)
    2. 1x 6-well: 0,5ml of virus from 01.09.2010 (testing R/C P325)
    3. 4x 6-well: 10x 0,5ml and 4x 1ml of virus from 01.09.2010 (stratagene R/C)
    4. 4x 6-well: 10x 0,5ml and 4x 1ml of virus from 04.09.2010 (10 plates same treatment)
    5. 6x 6-well: 0,5ml of virus from 21.08.2010 (TKGMK; 18x pH 7,10 and 6x pH 7,12)

    Repetition of Hybridisation and Cloning for Loop insertion BioBricks

    Investigator: Achim, Anna

    • Samples:

    1: 453_BAP
    11: 453_Z34C
    12: 587_Z34C
    13: 587_KO_Z34C
    14: 587_KO_Z34C_Spacer


    Comment: New approaches of the Hybridisation and Fill-in reactions were done because it didn't work the first time. A possible reason is that the klenow enzyme wasn't inactivated. In addition, the conditions for ligation will be improved. Also the ligation of 453_BAP was done again.

    Digestion:

    components Volume Vector 587 Volume Vector 453 Sample 11 Sample 12 Sample 13 Sample 14
    DNA 3,7 3,7 6 6 6 6
    BSA (10x) - - - - - -
    Buffer 4 (10x) 2 2 2 2 2 2
    Enzyme 1° Bam Ssp Ssp Bam Bam Bam
    Enzyme 2° Pvu Sal Sal Pvu Pvu Pvu
    H2O 12,3 12,3 10 10 10 10
    Total volume /µl20


    ° 1 µl each

    Preparation of gel:
    1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 50 minutes

    Repetition: Prepatation for SDM: cloning of Cap into pAAV

    Investigator: Stefan

    Schönen Abend dir noch

    Comment: On yesterday's plates nothing grew. Because gelex and ligation products were stored in coldroom, new ligation and trafo approaches were performed.



    Ligation approaches:

    • ligation with Quick Ligase (as performed yesterday)
    • ligation with T4 Ligase



    Transformation approaches:

    • Quick Ligase (approach from yesterday):
      • 2 µl of ligation product
      • 4 µl of ligation product


    • Quick Ligase (new approach):
      • 2 µl of ligation product
      • 4 µl of ligation product


    • T4 Ligase:
      • 2 µl of ligation product
      • 4 µl of ligation product


    Transformation:

    Trafo of each approach was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with ampicilin.

    Ligation and trafo of the modified Cap insert , the Viral Bricks 1, 11, 12, 13 and 14

    Investigator: Volker


    Ligation


    Construct Vector (µl) Insert(µl)
    ViralBrick 1 in 453 7.91 0.09
    ViralBrick 11 in 453 6.17 1.83
    ViralBrick 12 in 587 6.15 1.85
    ViralBrick 13 in 587 5.3 2.7
    ViralBrick 14 in 587 4.46 3.54
    p211 in pAAV-RC 5.52 3.54
    p211 in pAAV-RC 5.52 3.54



    Preparation of PBS Buffer

    Investigator: Volker

    Two liters of PBS buffer were prepared for the column purification of the viral particles. The following protocol was used:

    • dissolve the following in 800ml ddH2O:
      • 8g of NaCl
      • 0.2g of KCl
      • 1.44g of Na2HP0$ (1.69 because of the crystal water in the reagent)
      • 0.24 g of KH2PO4
    • adjust to pH 7.4
    • adjust to 1000ml
    • steril filtrate the solution

    111. labday 05.09.2010

    Mini-prep of glycerol stock pMA_RepCap Vector_SDM_InsPvuII clone 1 (B182)

    Investigator: Stefan

    Glycerol stock: Because glycerol stock B182 was not mixed before putting into -80 °C freezer the cells died. Therefore a new glycerol stock was prepared and labled B302.

    Mini-Prep was performed according to the standard protocol. Two preps were prepared:

  • P363 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (1) = 215,6 ng/µl
  • P364 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (2) = 230,5 ng/µl

    Picking clones of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

    Investigator: Hanna

    2 clones of each construct (1. pCerulean_Zegfr:1907_ShortLinker, 2. pCerulean_Zegfr:1907_MiddleLinker, 3. pCerulean_Zegfr:1907_LongLinker, 4. pCerulean_Zegfr:1907_SEG and 5. pCerulean_CFP_MiddleLinker) were picked.

    To do tomorrow (6.9.): Mini-Preps and Test digestion (with EcoRI and PstI).


    112. labday 06.09.2010

    Minipreps & Test PCR of pTert Biobrick assembly

    Investigator: Achim

    • After unsucessful test digestion with EcoRI and PstI I tried out a test PCR with Igem Primers (same Mastermix as for loop inserts)
    • Elongation time was set to 2.35 min because the amplified fragment should be about 2.5 kbp big.
    • Result:
    The visible bands are not specific. Either cloning or PCR went wrong :)
    • Next steps: Another test digestion tomorrow, if that doesn't work out I'll repeat the ligation.

    Minipreps & Test PCR of Loop inserts 1, 11, 12 ,13, 14

    Investigator: Achim & Anna

    • Samples:

    1: 453_BAP
    11: 453_Z34C
    12: 587_Z34C
    13: 587_KO_Z34C
    14: 587_KO_Z34C_Spacer


    • Minipreps of 1.1, 1.3, 11.1, 11.2, 12.1, 12.2, 13.1, 13.2, 14.1, 14.2
    • Clones 1.4, 11.3, 11.4, 12.3, 12.4, 13.3, 13.4, 14.3, 14.4 were pelleted and stored away in case the first two are incorrect.
    • In the hope to confirm our small inserts, A test PCR using Igem standard primers was carried out:

    PCR-reaction:

    Volume / µl ingredients
    2,5 Taq Buffer
    x template (~0.5 ng)
    2,5forward Igem primer (1:10)
    2,5 reverse Igem primer (1:10)
    0,5dNTP
    filled upt to 25H2O
    0,5Taq Polymerase (1.25)


    PCR program:

    Roundstemperature/ °CTime
    1 95 30"
    9525"
    25 61 30"
    68 50"
    1 68 5"


    Results:

    Bands should be about 150 for BAP and about 200 for Z34C. Bands dont quite match the expectations, somehow the BAP band seems bigger than the Z34C band. Clones 1.3, 11.1, 12.2, 14.1 were sent for sequencing.



    Mini-Preps and test digestion of Linker BioBricks

    Investigator: Anna

    Comment: Mini-preps of all clones were prepared. They have not been added to the excel sheets yet, but were stored in the freezer -> Samples of the usable and sequenced constructs were added to the excel sheets and stored in the freezer (06.09.10).


    Samples 1 2 3 4 5 6
    InsertVP1up_NLS_HisVP1up_NLS_His ZeGFP:1907_SEG ZeGFP:1907_SEG ZeGFP:1907_ML ZeGFP:1907_ML
    Clone12 1 2 1 2
    Concentration439,57463,51 415,24 354,5 406,75 382,27


    7 8 9 10 11 12
    CFP_ML CFP_MLZeGFP:1907_SLZeGFP:1907_SLZeGFP:1907_LLZeGFP:1907_LL
    1 2 1 2 1 2
    482,76424,76 497,06 413,55 391,05427,20

    SL: Shortlinker ML: Middlelinker LL: Longlinker

    Test digestion:

    components Volume for each sample /µl
    DNA 1
    BSA (10x) -
    Buffer 4 (10x) 1
    Enzyme EcoRI HF 0,5
    Enzyme Pst HF 0,5
    H2O 7
    Total volume /µl 10


    Preparation of gel:

    • Incubation time: 1 h, Incubation temperature: 37°
    • 1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes



    Freiburg10 pCerulean Linker 06.09.png

    • Expected fragment sizes:

    VP1up_NLS_Histag: 550 bp
    ZeGFP:1907_SEG: 344 bp
    ZeGFP:1907_ML: 261 bp
    CFP_ML: ~ 1000 bp
    ZeGFP:1907_SL: 249 bp
    ZeGFP:1907_LL: 237 bp

    To do: Test digestion of VP1up_NLS_mVenus and Affibody. Number 2, 3, 5, 7, 9 and 12 will be sent for sequencing tomorrow. For sequencing results of VP1up_NLS_Histag go to labday 09.09.2010, for the linker BioBricks go to labday 08.09.2010.

    Production of Ampicilin and Chloramphenicol

    Investigator: Jessica

    • 10ml ethanol (70%) containing 1g Amp in 60µl aliquots
    • 10ml ethanol (70%) containing 0,25g Cm in 60 µl aliquots


    Mini-prep and test digestion of pAAV_RC-CapIns_prepSDM

    Investigator: Stefan

    Mini-Prep was performed according to the standard protocol

  • P394 = pAAV_RC_CapIns_prepSDM clone 1 = 296,42 ng/µl
  • P395 = pAAV_RC_CapIns_prepSDM clone 2 = 274,92 ng/µl
  • P396 = pAAV_RC_CapIns_prepSDM clone 3 = 498,39 ng/µl
  • P397 = pAAV_RC_CapIns_prepSDM clone 4 = 487,67 ng/µl
  • P398 = pAAV_RC_CapIns_prepSDM clone 5 = 469,96 ng/µl
  • P399 = pAAV_RC_CapIns_prepSDM clone 6 = 517,06 ng/µl
  • P400 = pAAV_RC_CapIns_prepSDM clone 7 = 451,68 ng/µl
  • P401 = pAAV_RC_CapIns_prepSDM clone 8 = 479,18 ng/µl
    Constructs were digested with XcmI and Acc65I for 70 minutes at 37°C
    components pAAV_RC_CapIns_prepSDM clone 1-8
    DNA 2
    Buffer 2 (10x)1
    BSA (10x)1
    XcmI 0,5
    Acc65I 0,5
    H2O5
    Total volume 10



    Freiburg10 pAAV RC CapIns prepSDM.png


    Sent for sequencing: Clone 3 (P396) was sent for sequencing.

    • Tube name: SB1
    • Primer used: VP 4200 rev

    Sent for sequencing

    Investigator: Stefan

    • Tube name: SB2

    pAAV_RC_inserts clone1 (P326) was sent for sequencing using the following primers:

    • VP1 primer for pKex rev
    • SK
    • Primer Cap 2800 for
    • Primer Cap 2800 rev
    • Primer Cap 3500 for

    113. labday 07.09.2010

    Sequencing results of new loop insertion ligations

    Investigator: Achim

    • Sequencing confirmed the 453 BAP ligation contains the correct sequence. 453 Z34C and 587 Z34C didn't contain inserts, 587 KO Z34C still contained the BLA placeholder.

    Repetition of CD biobrick production

    Comment: Despite the results of the test digestion, the samples, which were sent for sequencing do not contain the GOI. Thus the whole procedure will be repeated today.

    Investigator: Kira
    DNA sample was diluted 1:100


    Ingredients CD sample
    5X Phusion HF buffer 10 µl
    10 mM dNTP mix1µl
    forward primer: O158 2,5µl
    reverse primer: O159 2,5 µl
    DNA Template0,5 µl
    DMSO 0 µl
    Phusion Polymerase0,5 µl
    H2O33µl
    Total volume50 µl


    PCR program:

    CyclesTemperatureTime
    98°C1
    8x98°C15"
    60°C25"
    72°C60"
    17x98°C15"
    65°C25"
    72°C60"
    1x72°C5'
    Hold 4°C


    Digestion of plasmid backbone:

    c (pSB1C3) = 151, 1 ng/ µl

    Components vector Volume/µL
    DNA 1 µg 6,0 µl
    BSA (100x) 0,2 µl
    Buffer no. 4 (10x) 2,0 µl
    Enzyme 1 XbaI 0,5 µl
    Enzyme 2 AgeI HF 0,5 µl
    H2O 10,8 µl
    Total volume 20


    incubation @ 37 C for approx. 2 h

    1% agarose gel

    Freiburg10 PCR product and digested pSB1C3 vector.jpg

    Digestion of PCR product:

    Components PCR product Volume/µL
    DNA 30,0 µl
    BSA (100x) 0,4 µl
    Buffer no. 4 (10x) 4,0 µl
    Enzyme 1 XbaI 1,5 µl
    Enzyme 2 AgeI HF 1,0 µl
    H2O 3,1 µl
    Total volume 40


    incubation @ 37 C for approx. 2 h

    Ligation

    Quickligase was used

    DNA-mix 9 ul (8,2 ul insert +0,8 ul insert)
    Quickligase 1ul
    Quickligase Buffer 10 ul

    Incubation @ RT for 10'

    Transformation

    BL21 cells were used for transformation because XL1B cells have to be tested first. Transformation according to the standard protocol.

    Comment: to figure out if, the digestion of the vector and the following ligation was successful, 2 plates have been prepared. One plate contains only the digestion vector, while the second contains the ligated sample.

    Test digestion of pCerulean_VP1up_NLS_mVenus and pCerulean_VP1up_NLS_Affibody

    Investigator: Anna


    Samples Insert Clone Concentration [ng/µl]
    1 VP1up_NLS_mVenus 1 416,59
    2 VP1up_NLS_mVenus 2387,46
    3 VP1up_NLS_Affibody 1390,68
    4 VP1up_NLS_Affibody 2374,12


    Test digestion:

    components Volume for each sample /µl
    DNA 2
    BSA (10x) -
    Buffer 4 (10x) 1
    Enzyme EcoRI HF 0,5
    Enzyme Pst HF 0,5
    H2O 6
    Total volume /µl 10


    Preparation of gel:

    • Incubation time: 2 h, Incubation temperature: 37°
    • 0,5 g Agarose, 50 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes



    Freiburg10 Test digestion VP1up NLS Affibody.jpg

    • Expected fragment sizes:

    VP1up_NLS_mVenus: 1215 bp
    VP1up_NLS_Affibody: ~ 700 bp

    Comment: Samples 1 and 3 were sent for sequencing. For sequencing results go to labday 09.09.2010.

    Reftrafo of pSB1A2_EGFP_HQ from distribution plate (Bba_I719015 plate 1 well 15P = T7GFPmut3bHQ)

    Investigator: Jessica
    P15 (T7GFPmut3bHQ) from plate 1 of the distribution plates from the iGEM headquarter was solved in 10 µl H2O

    • two trafos was prepared with 2 µl DNA and XL1blue (lab)
      • plate 1: 90µl DYT + 10µl mixed DNA-XL1b-solution
      • plate 2: resupended pellet


    Design and Ordering of GFP-Oligos

    Investigator: Hanna

    Comment: In order to perform single virus tracing experiments and in order to test the Affibody and the DARPin, we decided to use GFP which can be better visualized than CFP. Because we received GFP in the distribution kit we performed a trafo with the Bba_I719015. In addition to that we want to convert the GFP into the RFC25 standard - because for our purpose we want to be able to perform fusion approaches.
    Freiburg10 GFPPrimer.jpg

    Sequence analysis of pAAV_RC_CapIns_prepSDM

    Investigator: Stefan

    The synthetised Cap was inserted into the pAAV vector already containing the 4 mutations and the Rep construct.

    • Tube name: SB1
    • Primer used: VP 4200 rev



    Cloning of Cap into pAAV_RC_1.2 SDM SalI

    Investigator: Stefan

    Comment: pAAV with inserted Rep does not work in cell culture as expected. It was assumed that this is related to the deletion of a restriction site for KpnI at the splicing site of Rep40/Rep68. To test our synthethised Cap a plasmid only containing this will be produced.


    Digestion:

    components pMA_RepCap Vector_SDM_InsPvuII (P392) pAAV_RC_1.2 SDM SalI (P158)
    DNA 11,6 3,7
    Buffer 2 (10x)2 2
    XcmI 1 1
    BsiWI 11
    H2O4,4 12,3
    Total volume 20 20


    Comment:Digestion was performed using two steps, first incubating for 1 hour at 37 °C, afterwards for 1,5 hours at 55 °C.



    Gel:

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



    Freiburg10 4x mutant Cap.jpg




    Gelextraction:

    The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

    • pMA_RepCap Vector_SDM_InsPvuII (P392): c= 8,43 ng/µl
    • pAAV_RC_1.2 SDM SalI (P158) c= 17,24 ng/µl


    T4 Ligation:

    Comment: XcmI produces only 1 base overhang, but using Quick Ligase is not working properly. Therefore ligation is performed using T4.

    The Ligation was performed as following:

    • Vector Volume: 4,17 µl
    • Insert Volume: 3,83 µl


    • 1 µl T4 ligase buffer (10x)
    • 8 µl (Vector + Insert) mix
    • 1 µl T4 Ligase


    Incubating for 70 minutes.


    Transformation:

    Trafo was performed according to the standard protocol (BL21). The cells were plated on a agar plate with ampicilin.

    Repetition of Mini-Preps and test digestion of Linker BioBricks from 2nd ligation

    Investigator: Achim, Anna

    Comment: Recent update of succesfully produced ViralBricks:

    File:Freiburg10 Production of ViralBricks.pdf


    Test digestion:

    Number 11.4 11.4 12.3 12.4 13.3 13.4 14.3 14.4
    DNA76,5 5,5 5 5 12,52 8,57
    H2O5,56 7 7,57,5 0 45,5

    BSA: no
    Enzymes:
    EcoR I HF: 0,5 µl
    Pst I HF: 0,5 µl
    Total volume /µl:10

    • Incubation time: 3/4 h, Incubation temperature: 37°


    Preparation of gel:

    • 1 g Agarose, 50 ml TAE (2%), 6 µl GELRED , at 115 Volt, running time: 45 minutes



    Freiburg10 Freiburg10 07 09 ViralBricks.png

    To do: A new approach of the ViralBricks production has to be done tomorrow. A possible reason for the failure of the reactions is that the restriction enzymes didn't cut in the kenow buffer.

    Preparation for Midi-Preps of pAAV_mVenus, pHelper and pAAV_RC

    Investigator: Kerstin

    • 25 ml DYT was prepared with 25 µl Ampicillin and inoculated with glycerol stocks of pAAV_mVenus, pHelper and RC, each were incubated over night in 37°C room.


    Sequencing

    Investigator: Hanna
    pCerulean_Zegfr:1907_ShortLinker, pCerulean_Zegfr:1907_MiddleLinker, pCerulean_Zegfr:1907_LongLinker, pCerulean_Zegfr:1907_SEG and pCerulean_CFP_MiddleLinker were sent for sequencing.
    Used primer: GATC_std_CMV-F

    114. labday 08.09.2010

    Sequencing results of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

    Investigator: Hanna

    Comment: In order to perform single virus tracing experiments, CFP, which was fused to the Middle-Linker, was cloned into pCerulean. Referring to the establishment of a small linker library, the Affibody Zegfr:1907 was fused to the Short-Linker, Middle-Linker, Long-Linker and SEG-Linker and was then cloned into pCerulean.

    Sequencing results:
    1. pCerulean_CFP_MiddleLinker:
    Freiburg10 pCerulean CFP MiddleLinker.JPG

    Conclusion: Sequencing looked well, CFP_MiddleLinker was cloned successfully into pCerulean is now ready for fusion to VP2/3.

    2. pCerulean_Zegfr:1907_ShortLinker:
    Freiburg10 pCerulean Affi ShortLinker.JPG

    Conclusion: Sequencing looked also well, Zegfr:1907_ShortLinker was cloned successfully into pCerulean is now ready for fusion to VP2/3.

    3. pCerulean_Zegfr:1907_MiddleLinker:
    Freiburg10 pCerulean Affi MiddleLinker.JPG

    Conclusion: Sequencing looked also well, Zegfr:1907_MiddleLinker was cloned successfully into pCerulean is now ready for fusion to VP2/3.

    4. pCerulean_Zegfr:1907_LongLinker:
    Freiburg10 pCerulean Affi LL2.png

    Conclusion: Sequencing delivered that there was a C -> G transistion between the NgoMIV restriction site and the Zegfr:1907 sequence. Because it's located in the gene-synthesis region, the question is, whether there was a mutation caused by the XL1b cells or whether sequencing quality is not good enough. Therefore it was decided to sequence this conbstruct once again in reverse direction.

    5. pCerulean_Zegfr:1907_SEG:
    Freiburg10 pCerulean Affi SEG.JPG

    Conclusion: Sequencing looked also well, Zegfr:1907_SEG was cloned successfully into pCerulean is now ready for fusion to VP2/3.

    Repetition of PCR of SV40 terminator

    Investigator: Anna

    Comment: Second repetition of SV40 PCR, insert and vector were cut with Age I and Xba I.

    PCR sample: p230 (PeGFP_C1)
    Plasmid backbone: p51.2 (pSB1C3_CFP)


    Ingredients CD sample
    5X Phusion HF buffer 10 µl
    10 mM dNTP mix1µl
    forward primer: O158 2,5 µl
    reverse primer: O159 2,5 µl
    DNA Template3 µl
    DMSO 0 µl
    Phusion Polymerase0,5 µl
    H2O30,5 µl
    Total volume50 µl


    PCR program:

    CyclesTemperatureTime
    98°C1
    8x98°C15"
    59°C25"
    72°C7"
    17x98°C15"
    67°C25"
    72°C7"
    1x72°C5'
    Hold 4°C


    Digestion of plasmid backbone:

    c (pSB1C3_CFP) = 151, 1 ng/ µl

    Components vector Volume/µL
    DNA 1 µg 7,0 µl
    BSA (100x) 2,0 µl
    Buffer no. 4 (10x) 2,0 µl
    Enzyme 1 XbaI 0,5 µl
    Enzyme 2 AgeI HF 0,5 µl
    H2O 8 µl
    Total volume 20


    incubation @ 37 °C for approx. 2,5 h

    Preparation of gel:

    0,75 g Agarose, 50 ml TAE (1,5%), 6 µl GELRED , at 115 Volt, running time: 45 minutes
    Expected size of fragment: 261 bp

    Freiburg10 PCR SV40.jpg


    Digestion of PCR product:

    Components PCR product Volume/µL
    DNA 18,5 µl
    BSA (100x) 3 µl
    Buffer no. 4 (10x) 3 µl
    Enzyme 1 XbaI 1 µl
    Enzyme 2 AgeI HF 1 µl
    H2O 3,5 µl
    Total volume 30


    incubation @ 37 °C for approx. 2 h

    Ligation

    c(pSB1C3_cut_Age_Xba) = 0,9 ng/µl
    c(Insert) = 25,8 ng/µl

    T4 Ligase was used.

    DNA-mix 8 ul (7,08 µl insert + 0,92 µl insert)
    T4 Ligase 1µl
    T4 Ligase Buffer 1 µl

    Incubation @ RT for 20'

    Transformation

    Transformation was done according to the standard protocol with XL1B cells.

    PCR of P40

    Investigator: Stefan
    DNA sample was diluted 1:1000

    PCR progam:


    Ingredients Volume / µl
    5X Phusion HF buffer 10
    10 mM dNTP mix1
    forward primer: O152 2,5
    reverse primer: O153 2,5
    DNA Template1
    DMSO -
    Phusion Polymerase0,5
    H2O31,5
    Total volume50


    PCR program:

    CyclesTemperature / °CTime / s
    9860
    8x9815
    6125
    723
    17x9815
    6925
    724
    1x72300
    Hold 4


    Digestion of plasmid backbone:
    Plasmid used: pSB1C3_CFP (P51.2)
    c (pSB1C3_CFP) = 151, 1 ng/ µl

    Components vector Volume/µL
    DNA 10
    BSA (10x) 2
    Buffer no. 4 (10x) 2
    XbaI 1
    PstI 4
    H2O 4
    Total volume 20


    incubation @ 37 C for approx. 2 h


    Comment: PCR does not work out twice. Therefore troubleshouting will have to be done.

    Midi-Prep

    Investigator: Anissa, Kerstin

    Midi-Prep was performed with B34 (pAAV_iGEM_mVenus_YFP), R/C (standard P357) and pHelper (standard P356) according to the standard protocol.

    • c(pHelper): 518,7 ng/µl
    • c(RC): 1068,7 ng/µl
    • c(mVenus): 277,9 ng/µl


    Seeding HT1080, Passaging AAV293 and Transfection

    Investigator: Kerstin, Patrick

    • Seeding HT1080 cells for Transduction at 09.09.2010: two 6-well plates were prepared (200.000 cells per well)
    • Passaging AAV293 cells from T75 to T175 flasks
    • Transfection of AAV293: Ten plates same treatment (10µg of each plasmid):
      • c(RC)= 1068,7 ng/µl --> 9,4 µl
      • c(pHelper)= 518,7 ng/µl --> 19,3 µl
      • c(TKGMK, P81b)= 1900 ng/µl --> 5,3 µl

    Checking fractions of Äkta run

    Investigator: Stefan

    Comment: Fractions 6 - 11 of supernatant and of lysated cells treated with benzonase have to be checked for virus particles as well as fraction 20 of the second approach. This will be done amplifing the CMV promotor. Additionally, one positive control (pCerulean P274) and one negative control (HT1080 cells already confirmed being negative in qPCR) were used for PCR approach.


    PCR progam: primers used:

  • CMV_forward_qPCR
  • CMV_reverse_qPCR

    Ingredients Volume / µl
    10x ThermoPol buffer 10
    10 mM dNTP mix1
    forward primer: O20 2,5
    reverse primer: O21 2,5
    MgCl2 (50mM) 4
    fraction5
    DeepVent0,5
    H2O8
    Total volume25


    PCR program:

    CyclesTemperature / °CTime / s
    95600
    35x9520
    5620
    7218
    1x72120
    Hold 4


    Gel run will have to be performed tomorrow.

    115. labday 09.09.2010

    Picking clones of leftITR_hTERT_beta-globin and leftITR_CMV_beta-globin biobricks

    Kira
    The plates were checked in the morning and they did not contain any visible colonies, thus the plates were kept @ 37C till the late afternoon in order to check again.

    Fortunately, after approx. 18h of incubation the plate observation revealed lots of colonies, thus 3 clones from each plate could be inoculated in the DYT media.

    new ligation try and transformation of CD-biobrick

    Kira

    CD-biobrick plate contained just few (around 3 colonies), thus new ligation approach was performed. T4 ligase was used. The ligation mix was incubated @ 18C for around 5 hours

    Transformation was performed according to the standard protocol


    Repetition of ViralBrick Ligations: 453 Z34C, 587 Z34C, 587 KO Z34C, 587 KO Z34C Spacer

    Investigator: Achim

    • Nomenclature:

    11K: 453_Z34C in Klenow Buffer
    12K: 587_Z34C in Klenow Buffer
    13K: 587_KO_Z34C in Klenow Buffer
    14K: 587_KO_Z34C_Spacer in Klenow Buffer


    11 4: 453_Z34C in Buffer 4
    12 4: 587_Z34C in Buffer 4
    13 4: 587_KO_Z34C in Buffer 4
    14 4: 587_KO_Z34C_Spacer in Buffer 4

    Two new reaction setups for the remaining four ViralBricks:

    • Hybridisation & Fill-In reaction in Buffer 4, heat inactivation, digestion, ligation
    • Hybridisation & Fill-In reaction in Klenow Buffer, heat inactivation, PCR Purification, digestion, ligation

    We followed the recommendations provided by Fermentas with their Klenow Fragment manual:

    • 2 µl total DNA (3,3 µl per Oligo)
    • 0,1 µl dNTPs
    • 2 µl Klenow Buffer/ Buffer 4

    in a 20 µl reaction.

    Components 453 Z34C 587 Z34C 587 KO Z34C 587 KO Z34C SPACER
    O143O145O147O149
    O144O146O148O150

    Hybridisation was performed using the TKfill program on the gradient cycler. After Hybridisation, 0,5 µl Klenow fragment was added. The fill-in reaction was run for 40 minutes at 37°C. Afterwards, the Klenow fragment was heat inactivated at 75°C for 10 minutes. The samples containing the Klenow buffer were then purified with the QIAquick PCR Purification Kit Protocol.

    Samples 11 K and 11 4 were digested with SspI and SalI, Samples 12-14 were digested with BamHI and PvuII. Buffer 4 was added to the purified samples. One sample of the vector plasmid pSB1C3_BLA (P320) was digested with SspI and SalI, two samples were digested with BamHI and PvuII two obtain a sufficient amount of DNA for 6 ligations.

    (Table with concentrations and used DNA Volumes)

    After digestion, the digested inserts were separated on a 2% agarose gel, the vector backbones were separated on a 1% agarose gel:

    (Pictures of gels)

    Vektor 587
    Vektor 453
    Inserts


    (Nanodrop concentrations of gelex:)

    Ligation:

    (Ligation volumes)

    Vectors and Inserts were ligated for ~3h with T4 Ligase at room temperature.

    Two µl of each ligation were transformed into XL1B cells according to our standard protocol. Plates were incubated overnight

    Midiprep of pHelper, pAAV_RC and pAAV_iGEM_mVenus (B34)

    Investigator: Patrick
    The Midiprep was performed according to the standard protocol yieldung the following concentrations:

    pAAV_iGEM_mVenus (B34): 1131,7 ng/µl, labeled P419,420,421
    pHelper: 548 ng/µl
    pAAV_RC: 985 ng/µl

    Miniprep of pAAV_RC_CapIns_noRep and pSB1A2_EGFP_HQ

    Investigator: Stefan

    Mini-Prep was performed according to the standard protocol

  • P413 = pAAV_RC_CapIns_noRep clone1 c= 310,73 ng/µl
  • P414 = pAAV_RC_CapIns_noRep clone2 c= 217,66 ng/µl
  • P415 = pAAV_RC_CapIns_noRep clone3 c= 427,06 ng/µl
  • P416 = pSB1A2_EGFP_HQ clone1 c= 252,56 ng/µl
  • P417 = pSB1A2_EGFP_HQ clone2 c= 255,02 ng/µl
  • P418 = pSB1A2_EGFP_HQ clone3 c= 227,59 ng/µl
    components pAAV_RC_CapIns_noRep clone1-3pSB1A2_EGFP_HQ clone1-3
    DNA 1 1
    Buffer (10x) 1 1
    BSA (10x) 1 1
    Enzyme 1 Acc65I 0,5 XbaI 0,5
    Enzyme 2 BsiWI 0,5 PstI 0,5
    H2O 6 6
    Total volume 10 10



    Freiburg10 test digestion pAAV CapIns noRep pSB1A2 eGFP HQ.jpg



    Sent for sequencing: pAAV_RC_CapIns_noRep clone1 (P413)

    • Tube name: SB1
    • Primer used: VP 4200 rev

    Sent for sequencing: pSB1A2_EGFP_HQ clone1 (P416)

    • Tube name: SB2
    • Primer used: VR2

    PCR of VP2/VP3

    @jessi: Did you clone the constructs theoretically?? I cannot find it? And is there any special reason that you used the pSB1C3_SEG for inserting the PCR products? And i just saw that u digested the PCR product with PstI, is that just a mistake happend accidentally by writing it down?? Thanks a lot!(Bea)

    Comment: VP2/3 will be separated from cap for n-terminal fusion

    Investigator: Jessica
    DNA sample was diluted 1:1000

    • P357 = pAAV_RC
    • P396 = pAAV_RC_CapIns_prepSDM clone 3

    PCR progam:


    Ingredients Volume / µl Volume / µl
    5X Phusion HF buffer 10 10
    10 mM dNTP mix1 1
    forward primer: O90 2,5 2,5
    reverse primer: O120 2,5 2,5
    DNA Template0,78 2
    DMSO 1 1
    Phusion Polymerase0,5 0,5
    H2O31,72 30,5
    Total volume50 50


    PCR program:

    CyclesTemperature / °CTime / s
    9860
    8x9815
    6125
    7230
    17x9815
    7225
    7230
    1x72300
    Hold 4


    Digestion of plasmid backbone:
    Plasmid used: pSB1C3_RFC_SEG (P107)
    c (pSB1C3_RFC_SEG) = 185,6 ng/ µl

    Components vector Volume/µL
    DNA 5,4
    BSA (10x) 2
    Buffer no. 4 (10x) 2
    NgoMIV 1
    SpeI 1
    H2O 9,6
    Total volume 20


    incubation @ 37 C for approx. 2h

    1% agarose gel

    Digestion of PCR product:

    Components PCR product Volume/µL
    DNA 30
    BSA (10x) 4
    Buffer no. 4 (10x) 4
    NgoMIV 1
    SpeI 1
    H2O -
    Total volume 40


    incubation @ 37 C for approx. 2,5h

    Ligation:

    1 µl T4 DNA ligase, 1 µl 10x Buffer, µl vector (P107), µl Insert (P357)/ µl Insert (P396).
    Incubation time: over night

    Sequencing results of pCerulean_VP1up_NLS_Affibody/mVenus/Histag

    Investigator: Anna

    Comment: All results were classified in agreement with Sven. They can be used for generating the full construct (with VPex3).


    pCerulean_VP1up_NLS_Affibody: Insert ok, but there is a G -> C transition.
    Freiburg10 pCerulean VP1up NLS Affibody.JPG

    pCerulean_VP1up_NLS_mVenus: The NgoMIV restriction site is present, but as above, there is a G -> C transition. Third mutation downstream is a sequencing fault, there are clearly two peaks for C.
    Freiburg10 pCerulean VP1up NLS mVenus.JPG

    pCerulean_VP1up_NLS_Histag: Insert was succesfully cloned into p_Cerulean.
    Freiburg10 pCerulean VP1up NLS Histag.JPG

    Biobrick production of leftITR-phTERT-beta-globin and leftITR_pCMV-beta-globin

    Motivation: Production of fusion proteins

    Investigator: Kira

    Digestions of backbones and inserts: c (pSB1C3_leftITR_phTERT) = 179 ng/ µl
    c(pSB1C3_leftITR_pCMV) = 231 ng/ul

    Components TERT CMV beta-globin
    DNA 5,5 4,5 8,5
    BSA (100x) 0,25 0,25 0,25
    Buffer no. 4 (10x) 2,5 2,5 2,5
    PstI-HF 0,5 0,5 0,5
    SpeI 1 1 1
    XbaI 0 0 1
    H2O 15,25 16,25 12,25
    Total volume 25 25 25


    incubation @ 37 C for approx. h

    1% agarose gel

    Freiburg10 hTERT& CMV biobricks 2010 09 10.jpg


    Ligation:

    Quickligase was used

    TERT-mix 9 ul (5,3 ul vector + 3,7 ul insert)
    CMV-mix 9 ul (3,7 ul vector + 5,3 ul insert)

    Quickligase 1ul
    Quickligase Buffer 10 ul

    Incubation @ RT for 10'

    Transformation:
    Transformation was performed according to the standard protocol w XL1B cells (thanks Achim!)

    Transfection, Harvest viral particles and Transduction

    Investigator: Kerstin

    • Transfection of 15x 6-well plates (each plate same treatment --> 10 µg each plasmid)
    • Harvest viral particles (from 06.09.2010) and Transduction of HT1080 cells

    1.plate:

      • 1.1 no virus
      • 1.2 lITR_CMV_beta-globin_mVenus_rITR
      • 1.3 lITR_CMV_beta-globin_mVenus_rITR
      • 1.4 lITR_CMV_beta-globin_mVenus_rITR
      • 1.5 lITR_CMV_beta-globin_mVenus_Hgh_rITR
      • 1.6 lITR_CMV_beta-globin_mVenus_Hgh_rITR

    2.plate:

      • 1.1 no virus
      • 1.2 lITR_CMV_mVenus_Hgh_rITR
      • 1.3 lITR_CMV_mVenus_Hgh_rITR
      • 1.4 lITR_CMV_mVenus_Hgh_rITR
      • 1.5 lITR_CMV_beta-globin_mVenus_Hgh_rITR
      • 1.6 lITR_CMV_beta-globin_mVenus_Hgh_rITR

    Competent XL1-B cells

    Investigator: Patrick

    Mistake.png

    Just for you! ;-)Do you know the normal transformation efficiency of chemically competent cells? What is good, waht is bad?? Thanks!

    Today i checked the mutual competent cells i made for their transformation ability. It is ok (>10^6 cells / µg DNA).
    Now there are about 100 new aliquots (each 100 µl) ready to use.

    Checking PCR amplified virus particles

    Investigator: Bea

    Comment: The virus particles purifed by using liquid chromatography ("ÄKTA")(containing which construct??) were PCR- amplified (dame primers were used as for the qPCR which were already tested and could be confirmed for proper working) in order to obtain insights into which collected fraction does contain our virus particels. Therefore the PCR amplified samples were loaded on an 2% agarose gel.

    • Virus particle containing following plasmid: pAAV_iGEM_mVenus
    • Liquid chromatography performed by: Volker
    • PCR amplification performed by: Stefan


    Results:

    Sorry mistake: The blue highlighted boxes represent the cell pellet, not the supernatant!


    As it can be seen in the picture, the expected PCR-fragment size of 200 bp can be detected in each fraction. Fractions collected 6 to 11 of the supernatant after cell lysis and centrifugation and fractions collected 6 to 11 of the cell pellet after cell lysis and centrifugation. The fraction 20 of the cell pellet was aswell PCR amplified and loaded on the agarose gel. The postivie control (added the plasmids containing a CMV promoter which were sequenced and confirmed) is loaded (pCerulean P274) and the negative control which contained only harvested HT1080 cells (harvested, but whithout any transduction) can be seen in lane 16 and 17, respectively. Since the PCR has been performed with a low TM and 35 cycles this could be the reason for the detection of the CMV promoter of the negative control HT1080. next time PCR should be carried out with only 25 cycles and a higher TM= 60°C. But the detection of the CMV promoter by using a normal PCR does work.

    Quickchange site directed mutagenesis of pAAV_RC_CapIns_prepSDM

    Investigator: Stefan

    Comment: In cellculture, pAAV with inserted synthesised Rep did not work out. It was suggested that this could be related to an insertion of a KpnI restriction site near a splicing site. To verify this, the restriction site needs to be removed and tested in cellculture again.


    Ingredients:


    Ingredients Volume / µl
    10x reaction buffer 2,5
    dNTP0,5
    forward primer: O182 0,52
    reverse primer: O183 0,52
    DNA Template2
    QuikSolution Reagent0,75
    QuikChangeLightning Enzyme0,5
    H2O17,71
    Total volume25


    PCR program:

    CyclesTemperature / °CTime / s
    95120
    8x (step 2-49515
    6010
    68225
    68600


    After PCR, the product was incubated with DpnI at 37 °C for 10 minutes.


    Transformation:

    Transformation was performed according to standard protocol using XL10-Gold cells. Cells were plated on plate containing Ampicilin.

    116. labday 10.09.2010

    Trafo evaluation of CD-biobrick plate

    Investigator: Kira

    Compared to the last tries, the ligation with T4 ligase was much more successful. The plate containes colonies.

    Picking clones of yesterdays ViralBrick ligations and of Jessicas ligation

    Investigator: Achim


    Seeding HT1080 cells

    Investigator: Kerstin

    • Ten 6-well plates of HT1080 cells were seeded (Transduction at 11.09.2010)for testing the optimal concentration of ganciclovir

    Test digestion of pSB1C3_SV40_Terminator_Clone1-3

    Investigator: Anna


    Samples Insert Clone Concentration [ng/µl]
    1 pSB1C3_SV40_Terminator 1 204,2
    2 pSB1C3_SV40_Terminator 2246,1
    3 pSB1C3_SV40_Terminator 3200,9


    Test digestion:

    components Volume for each sample /µl
    DNA 3
    BSA (10x) 1
    Buffer 4 (10x) 1
    Enzyme HF 0,5
    Enzyme HF 0,5
    H2O 4
    Total volume /µl 10


    • Incubation time: 1,5 h, Incubation temperature: 37°

    Preparation of gel:

    • 0,5 g Agarose, 50 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 40 minutes

    Freiburg10 Test digestion SV40.jpg

    • Comment: As shown on the gel, the vector backbone was religated with CFP (size: ~730 bp). PCR and Cloning steps have to be repeated, because the wrong restrition enzymes were used.


    Sequencing analysis of pSB1A2_eGFP_HQ and pAAV_RC_CapIns_noRep

    Investigator: Stefan
    pSB1A2_eGFP_HQ

    Comment: To improve iGEMs eGFP, it will be converted into RFC25 Freiburg standard. Plasmid preped from retrafo was sent for sequencing to verify sequence.


    Freiburg10 Sequencing eGFP.jpg


    pAAV_RC_CapIns_noRep

    Comment: Since pAAV containing the synthetised Rep does not work out in cell culture an additional approach was performed inserting only the synthesized Cap and test this.

    Freiburg10 Sequencing pAAV RC CapIns noRep.jpg

    Mini-prep and test digestion of leftITR_hTERT_beta-globin and leftITR_CMV_beta-globin biobricks

    Investigator: Kira

    Mini-Prep was performed with 3 clones of each construct in order to continue with test digestion and sequencing.

    Test digestion:

    components Volume for each sample /µl
    DNA 2.5
    BSA (10x) 2
    Buffer 4 (10x) 2
    Enzyme Xba 1
    Enzyme PstI-HF 0,5
    H2O 12
    Total volume /µl 20

    1% agarose gel

    Freiburg10 test digestion of hTERT& CMV biobricks 2010 09 10.jpg

    CMV_clone1 as well as hTERT_clone1 were sent for sequencing

    117. labday 11.09.2010

    Sequencing evaluation of leftITR_CMV_beta-gloin and leftITR_hTERT_beta-globin biobricks

    Investigator: Kira

    The sequencing reveals that both biobricks were seccessful cloned.

    Comment: For the evaluation, beta-globin intron was extracted from the original beta-globin biobrick and checked against the sequencing data (no connection to the lab server at home. the 'single' biobricks are only data, which are saved on my mac)

    Freiburg10 sequencing 2010 09 10.jpg

    FACS analysis, harvest viral particles and Transduction

    Investigator: Kerstin

    • FACS analysis of Constructs with/without Hgh/ß-globin
    • Harvested viral particles of transfection from 08.09.2010
    • Transduction for testing the optimal concentration of ganciclovir

    Mini-Preps of Z34C ViralBricks

    Investigator: Anna

    Recent update of ViralBricks: The hybridisation and fill-in reactions of the Z34C constructs were repeated with heat inactivation of the Klenow enzyme (see labday 04.09.10), without usable results. In addition, a second ligation of 453_Bap was done with succes. This time two new approaches of the fill-ins were prepared (labday 09.09.10), one by using the Klenow Buffer and one with the Standard NEB Buffer 4.


    Buffer 4:

    Samples Insert Clone Concentration [ng/µl]
    11.4.1 453_ Z34C 1 271,67
    11.4.2 453_ Z34C 2 218,01
    12.4.1 587_Z34C 1 199,88
    12.4.2 587_Z34C 2 357,13
    - 587_KO_Z34C--
    - 587_KO_Z34C--
    14.4.1 587_KO_Z34C_Spacer1 172,76
    14.4.2 587_KO_Z34C_Spacer2 337,54


    Klenow Buffer:

    Samples Insert Clone Concentration [ng/µl]
    11.1 K 453_ Z34C 1 220,89
    11.2 K 453_ Z34C 2 176,34
    12.1 K 587_Z34C 1 198,73
    12.2 K 587_Z34C 2 182,64
    13.1 K 587_KO_Z34C1186,50
    13.2 K 587_KO_Z34C2158,51
    14.1 K 587_KO_Z34C_Spacer1 182,25
    14.2 K 587_KO_Z34C_Spacer2 180,06


    Mini-Preps of separated VP2/3 for N-terminal fusion

    Investigator: Anna

    • P357 = pAAV_RC
    • P396 = pAAV_RC_CapIns_prepSDM clone 3


    Samples Clone Concentration [ng/µl]
    357.1 1 290,74
    357.2 2 282,97
    357.3 3 307,63
    396.1 1 302,04
    396.2 2 314,58
    396.3 3 298,77


    Update and next steps of daily labwork

    Investigators: Bea, Stefan

    Preparation of pAAV_RC construct

    1. We inserted the synthesized “rep” gene into the pAAV_RC construct with the four mutated restriction sites PstI (positions 310 and 4073), SalI (position 1239) and BamHI (position 856). The construct was tested in cell culture and no YFP expression of the vectorplasmid could be detected. Therefore, several new approaches were conducted.
      1. The first approach was to remutate the only “unwanted” restriction site(KpnI) in the supposed regulatory region of the rep gene. The basic idea was that we delete the KpnI site because KpnI was in the 453 loop in which we could insert additional small motifs. But, this idea was dismissed after some more research. Therefore the KpnI restriction site does not have to be deleted in the rep sequence. This construct will be tested in cell culture.
      2. Second, the experiment conducted in cell culture will be repeated with the same construct in order to exclude errors and mistakes related to the realization in cell culture.
      3. Since we cannot be sure if the inserted rep containing the remutated KpnI restriction site still works in cell culture, we decided to insert the synthesized “cap” into the already tested and confirmed pAAV_RC containing only the four mutated restriction sites (see above). This construct will be tested in cell culture aswell.


    Overview:

    no plasmid number yet P158 P250 P396 P413
    pAAV_RC (4fold mutated, ins rep, SDM KpnI, inserted “cap”) pAAV_RC (4-fold mutated) P158 pAAV_RC (4fold. Inserted “rep”)P250 pAAV_RC (4fold mutated, rep, cap)P396 pAAV_RC ((4fold mutated, cap) P413
    not yet tested in cell culture tested in cell culture and works tested in cell culture, does not work. Experiment will be repeated will not be tested in cell culture -> was used for SDM in rep gene. has to be tested P413
    desired construct. Clones were picked today!! contains many restriction enzymes. Is not the construct of choice If second experiment fails again, will not be used then. was only used for SDM in rep gene. If cell culture experiments work --> synthesized cap still functions properly.



    N-terminal fusion

    The constructs for the VP1 insertion and the Vp-2 fusion are ready for the next cloning step which means that we only need to fuse the VP2/3 PCR construct to the engineered plasmids containing different approaches. Two strategies will be followed until now:

    1. The first strategy is to PCR amplify the VP2/3 construct from the pAAV_RC (4fold mutated, P357 in order to fuse the working VP2/3 contruct to the targeting approaches.

      was performed by Jessica (9.9.10), waiting for results

    2. The second strategy is to use the pAAV_RC (4fold mutated, ins rep, cap, P396) to PCR amplify the desired construct VP2/3.

      was performed by Jessica (9.9.10), waiting for results

    3. The next step is to PCR amplify the VP2/3 aswell from the construct pAAV_RC (4fold mutated, ins rep, SDM KpnI, inserted “cap”)

      plasmid should be ready tomorrow (12.9.10), can be performed afterwards

      Cartoons48.jpg

      Comment (Hanna): We want to amplify VP2/3 from pAAV_RC (it's indifferent, whether the PCR is done with the "WT"-pAAV_RC or with the pAAV_RC- 4fold mutated... (the mutations are in rep!) Further on, we want to PCR amplify VP2/3 from pAAV_RC_capins and from pAAV_RC_capIns_HSPG-KO!!! Did you mean that?

      Yes you are right! Thanks! We should keep that in mind, and do not forget about the HSPG knockout!!! (Bea) ;-)

    Additionally, a site-directed mutagenesis has to be performed in order to obtain the plasmids which can be co-transfected with the modified construct containing the targeting molecules. Aim is, to knockout VP1 for VP1-insertion approaches and VP2 (knockout) for VP-2 fusion. This site-directed mutagenesis has to be performed at several constructs:

    1. P357 --> pAAV_RC (4fold mutated)

      will be performed tomorrow by Bea/Stefan (12.9.10)

    2. P396 --> pAAV_RC (4fold mutated, ins rep, cap)

      will be performed tomorrow by Bea/Stefan (12.9.10)

    3. P413 --> pAAV_RC (4fold mutated, ins cap)

      will be performed tomorrow by Bea/Stefan (12.9.10)

    4. pAAV_RC (4fold mutated, ins rep, SDM KpnI, inserted “cap”) --> since no Mini-preps have been prepared until now, no plasmid number is available.

      dependant on test digestion performed tomorrow (12.9.10), if it works out, it will be performed tomorrow (12.9.10) as well

    118. labday 12.09.2010

    Mini-Prep and test digestion of pAAV_RC_RepCapIns_SDMKpnI

    Investigator: Stefan

    Glycerol stocks were prepared:

  • B371 = pAAV_RC_RepCapIns_SDMKpnI clone 1
  • B372 = pAAV_RC_RepCapIns_SDMKpnI clone 2
  • B373 = pAAV_RC_RepCapIns_SDMKpnI clone 3


    Mini-Prep was performed according to the standard protocol

  • P431 = pAAV_RC_RepCapIns_SDMKpnI clone 1 = 362,21 ng/µl
  • P432 = pAAV_RC_RepCapIns_SDMKpnI clone 2 = 344,49 ng/µl
  • P433 = pAAV_RC_RepCapIns_SDMKpnI clone 3 = 331,30 ng/µl


    Constructs were digested with KpnI for 70 minutes at 37°C, as controls the following constructs were used:
  • pAAV_RC_1.2SDMSalI (P158)
  • pAAV_RC_CapIns_prepSDM (P396)
  • pAAV_RC_CapIns_noRep (P413)
    components pAAV_RC_RepCapIns_SDMKpnI clone 1-3 pAAV_RC_1.2SDMSalI (P158) pAAV_RC_CapIns_prepSDM (P396) pAAV_RC_CapIns_noRep (P413)
    DNA 1 1 1 1
    Buffer 1 (10x)1 1 1 1
    BSA (10x)1 1 1 1
    KpnI 0,5 0,5 0,5 0,5
    H2O6,5 6,5 6,5 6,5
    Total volume 10 10 10 10


    Preparation of gel:

  • 0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes

    Freiburg10 test digestion pAAV RC RepCapIns SDMKpnI.jpg

    Comment: Test digestions look fine. Work will be continued with clone 2 (P432) which will be sent for sequencing tomorrow as well.

    Test digestion of pSB1C3_VP2/3 needed for N-terminal fusion

    Investigator: Bea

    Comment: Test digestion of the two different approaches Jessica started on Friday has been performed. The general idea of the approach is to obatain VP2/3 cap genes (which means that the 137 amino acids of VP1 are missing) which then can be fused to the targeting molecules for the VP1 insertion and VP3 fusion approaches.
    Note: P257_1 - 3 means that the pCR has been carried out with this construct. Same can be pbserved with the plasmid number P296.

    Test digestion has been carried out using following enzymes:

    • Enzyme 1: PstI-HF
    • Enzyme 2: NgomIV


      Mastermix/µL P357_1/µL P357_2/µL P357_2/µL P396_1/µL P396_2/µL P396_2/µL
    DNA - 2 2 2 2 2 2
    BSA (10x) - 2,5 2,5 2,5 2,5 2,5 2,5
    Buffer 4 (10x) 10,5
    Enzyme NgoMIV 3,5
    Enzyme PstI 3,5
    H2O - 10,5 10,5 10,5 10,5 10,5 10,5
    Total volume 15 15 15 15 15 15 15


    • Test digestion was carried out for 55 minutes at 37°C.
    • The samples were loaded on a 1% agarose gel

    Loading plan:

    M   P357_1   P357_2   P357_3   P396_1   P396_2   P396_3 
    


    Results:

      Expected sizes of the two approaches has been:
      • "P357" = pSB1C3_RC: 304bp , 1651bp , 2061bp
      • "P396"= pSB1C3_RC_ins_rep and cap: 1951bp , 2061bp


    Freiburg10 Test digest pSB1C3 VP2-3 12.09.10.png

    Quickchange site directed mutagenesis of pAAV_RC: VP1/VP2 knockout

    Investigator: Stefan

    Comment: A site-directed mutagenesis has to be performed in order to obtain the plasmids which can be co-transfected with the modified construct containing the targeting molecules. Aim is to knockout VP1 for VP1-insertion approaches and VP2 (knockout) for VP-2 fusion. This site-directed mutagenesis has to be performed at several constructs:


    Knock-out of VP1:

  • pAAV_RC_1.2SDMSalI (P158)
  • pAAV_RC_CapIns_prepSDM (P396)
  • pAAV_RC_CapIns_noRep (P413)
  • pAAV_RC_RepCapIns_SDMKpnI (P432)

    Knock-out of VP2:

  • pAAV_RC_1.2SDMSalI (P158)
  • pAAV_RC_CapIns_prepSDM (P396)
  • pAAV_RC_CapIns_noRep (P413)
  • pAAV_RC_RepCapIns_SDMKpnI (P432)

    Used primers:
    Knock-out of VP1:
  • primer forward: VP1-M1L_for (O170)
  • primer reverse: VP1-M1L_rev (O171)
    Knock-out of VP2:
  • primer forward: VP2-ko_for (O165)
  • primer reverse: VP2-ko_rev (O166)

    Used plasmid and water per approach:
    plasmid no used plasmid (same for both approaches) (1:100) / µl VP1-ko: used water / µl VP2-ko: used water / µl
    P1580,518,9919,23
    P396217,4917,73
    P413316,4916,73
    P432316,4916,73


    Ingredients:

    Ingredients VP1-ko approach Volume / µl VP2-ko approach Volume / µl
    10x reaction buffer 2,5 2,5
    dNTP0,5 0,5
    forward primer: O170 - 0,38 O165 - 0,50
    reverse primer: O171 - 0,38 O166 - 0,52
    DNA Templatesee above see above
    QuikSolution Reagent0,75 0,75
    QuikChangeLightning Enzyme0,5 0,5
    H2Osee above see above
    Total volume25 25


    PCR program:

    CyclesTemperature / °CTime / s
    195120
    2 8x(step 2-4)9515
    36010
    468225
    568600


    After PCR, the products were incubated with DpnI at 37 °C for 10 minutes.


    Transformation:

    Transformation was performed according to standard protocol using XL10-Gold cells. Cells were plated on plate containing Ampicilin.

    Comment: There were not enough cells for all approaches. Therefore, only transformation of approach 5 and 8 were performed using 45 µl of cells. The other approaches were conducted with 30 µl.



    119. labday 13.09.2010

    Biobrick assembly: Cloning VP2/3 and VP2/3_ins_Cap to several targeting approaches

    Investigator: Stefan

    Comment: Goal of the experiment is to fuse VP2/3 or VP2/3_ins_Cap to pCerulean_VP1up_NLS_Affibody, pCerulean_VP1up_NLS_ Histag and pCerulean_VP1up_NLS_mVenus.

    Digestion of the constructs:
    Inserts:

    • pSB1C3_VP2/3 (P440) c= 290,74 ng/µl
    • pSB1C3_VP2/3_ins_cap (P444) c= 314,58 ng/µl

    Vectors:

    • pCerulean_VP1up_NLS_Affibody (P422) c= 390,68 ng/µl
    • pCerulean_VP1up_NLS_Histag (P424) c= 439,57 ng/µl
    • pCerulean_VP1up_NLS_mVenus (P426) c= 416,59 ng/µl


    Restriction enzymes:
    The inserts were digested using SpeI and NgoMIV, the vectors were digested using AgeI and SpeI.

    Components P440 /µL P444 /µL P422 /µL P424 /µL P426 /µL
    DNA 54,56,55,56
    BSA (10x) 2 2 2 2 2
    Buffer 4 (10x) 2 2 2 2 2
    Enzyme 1 1 1 1 1 1
    Enzyme 2 1 1 1 1 1
    H2O 9 9,5 7,5 8,5 8
    Total volume 20 20 20 20 20


    Gel:

    Comment: Hungry and tired. Picture will be added tomorrow...


    T4 Ligation:

    All ligation approaches were performed using 1 µl of vector and 7 µl of insert. 1 µl of T4 ligation buffer and 1 µl of T4 ligase were used.

    Transformation:

    Transformation was performed according to standard protocol using BL21 cells and plates containing Kanamycin.

    Biobrick production: P40 & SV40

    Investigator: Patrick

    So did u repeat the experiment?? Or are u ging to repeat it tomorrow?? (You know who I am ;-)


    P158 (398 ng/µl) was used as P40 template and P230 (367 ng/µl) as SV40 template. Both of them were diluted 1:400.

    SV40:


    Ingredients volume
    5X Phusion HF buffer 10 µl
    10 mM dNTP mix1µl
    forward primer: O158 2,5 µl
    reverse primer: O159 2,5 µl
    DNA Template1 µl
    DMSO 0 µl
    Phusion Polymerase0,5 µl
    H2O32,5 µl
    Total volume50 µl


    CyclesTemperatureTime
    98°C1
    8x98°C15"
    59°C25"
    72°C7"
    17x98°C15"
    67°C25"
    72°C7"
    1x72°C5'
    Hold 4°C



    P40:


    Ingredients Volume / µl
    5X Phusion HF buffer 10
    10 mM dNTP mix1
    forward primer: O152 2,5
    reverse primer: O153 2,5
    DNA Template1
    DMSO -
    Phusion Polymerase0,5
    H2O32,5
    Total volume50


    PCR program:

    CyclesTemperature / °CTime / s
    9860
    8x9815
    6125
    7210
    17x9815
    6925
    7210
    1x72300
    Hold 4


    Expected size of the PCR products: P158/p40: 180 bp, P230/SV40: 230 bp.

    Freiburg10 patrick p40 sv40 modified2.jpg

    The visible bands do not have the expected size. These are probably amplified primers. The experiment has to be repeated.

    Tobi proposed the following changes:

    Use 1,5 ng template for p40 and SV40.

    p40 PCR program:

    CyclesTemperature / °CTime / s
    9830
    10x9810
    5825
    7210
    15x9810
    6625
    7210
    1x72300
    Hold 4



    SV40 PCR program:

    CyclesTemperatureTime
    98°C30"
    10x98°C10"
    56°C25"
    72°C10"
    15x98°C10"
    64°C25"
    72°C10"
    1x72°C5'
    Hold 4°C


    Test digestion of ViralBrick ligation and pTert BioBrick assembly

    Investigator: Achim, Anna

    • Our Samples were digested with EcoRI and PstI again.
    • We used negative controls pSB1C3_Bla, containing a ~800bp insert and pSB1C3_SEG_linker, containing a ~160bp insert.
    • Additionally, a new test digestion of the pTert Biobrick assembly was prepared,this time with DrdI, an enzyme that should cut once in the insert and once in the vector. Expected sizes: ~2000 & 2500bp
    Freiburg10 Test Digestion Achim.PNG

    PCR of RepCap/Cap

    Investigator: Jessica
    DNA sample was diluted 1:100

    • P158 = pAAV_RC_1.2 SDM SalI
    • P396 = pAAV_RC_CapIns_prepSDM clone 3
    • P413 = pAAV_RC_CapIns_noRep clone 1
    • P432 = pAAV_RC_RepCapIns_SDMKpnI clone 2

    PCR progam:
    for Rep Cap

    Ingredients Volume P158 / µl Volume P396/ µlVolume P413/ µlVolume P432/ µl
    5X Phusion HF buffer 10 101010
    10 mM dNTP mix1 111
    forward primer: O93 2,5 2,52,52,5
    reverse primer: O120 2,5 2,52,52,5
    DNA Template0,250,200,320,30
    DMSO - ---
    Phusion Polymerase0,5 0,50,50,5
    H2O33,25 33,3033,1833,20
    Total volume50 505050


    no DNA on the gel...

    for Cap

    Ingredients Volume P158 / µl Volume P413/ µl
    5X Phusion HF buffer 10 10
    10 mM dNTP mix1 1
    forward primer: O93 2,5 2,5
    reverse primer: O120 2,5 2,5
    DNA Template0,250,32
    DMSO - -
    Phusion Polymerase0,5 0,5
    H2O33,25 33,18
    Total volume50 50


    PCR program:

    CyclesTemperature / °CTime / s
    9860
    8x9815
    6025
    7270
    17x9815
    6925
    7270
    1x72300
    Hold 4


    Digestion of plasmid backbone:
    Plasmid used: pSB1C3_VCK_Bla (P320)
    c (pSB1C3_VCK_Bla) = 408,0 ng/ µl

    Components vector Volume/µL
    DNA 2,45
    BSA (10x) 2
    Buffer no. 4 (10x) 2
    NgoMIV 1
    SpeI 1
    H2O 11,55
    Total volume 20


    incubation @ 37 C for approx. 2h

    1% agarose gel

    Digestion of PCR product:

    Components PCR product Volume/µL
    DNA 30
    BSA (10x) 4
    Buffer no. 4 (10x) 4
    NgoMIV 1
    SpeI 1
    H2O -
    Total volume 40


    incubation @ 37 C for approx. h

    Ligation:

    1 µl T4 DNA ligase, 1 µl 10x Buffer, µl vector (P320), µl Insert (P158)/ µl Insert (P413).
    Incubation time:

    Miniprep and test digestion of CD biobricks

    Investigator: Kira

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


    incubation @ 37 C for approx. 2h

    1% agarose gel

    BioBrick TM assembly of pSB1C3_leftITR_promoter_betaglobin to several GOIs

    Investigator: Bea

    Comment: Since we only assembled the vecotrplasmid with the CMV promoter and mVenus_YFP as gene of interest other approaches have to be conducted, especially the vectorplasmid which contains the tumorspecific promoter phTERT with the gene of interest mGMK_TK30. These approaches were started by Kira some days ago, and will now be continued. This time we are fusing the extisting and confirmed constructs pSB1C3_leftITR_CMV_betaglobin and pSB1C3_leftITR_phTERT_betaglobin to the gene of interests mVenus_YFP and mGMK.


    Digestion of the constructs:

    • P434 = pSB1C3_leftITR_CMV_betaglobin c=331ng/µL
    • P434 = pSB1C3_leftITR_phTERT_betaglobin c=331ng/µL


    Components vP434 /µL vP437 /µL
    DNA 4,54,5
    BSA (10x) 2 2
    Buffer no. 4 (10x) 2 2
    SpeI 1 1
    PstI-HF 1 1
    H2O 9,5 9,5
    Total volume 20 20


    Digestion of the three targeting approaches:

    • P81 = pAAV_iGEM_mGMK_TK30 --> approach will not be proceeded becuase the iGEM restriction sites cut in the vctor as well and therefore the desired fragments cannot be isolated with used iGEM restriction sites.!!!
    • P195 = pSB1C3_mGMK c=258ng/µL
    • P229 = pAAV_iGEM_mVenus c=486 ng/µL


    Components vP81 /µL vP195 /µL vP229 /µL
    DNA 7,8 12,3 4,1
    BSA (10x) 2,5 2,5 2,5
    Buffer no. 4 (10x) 2,5 2,5 2,5
    XbaI 1 1 1
    PstI-HF 1 1 1
    H2O 10,2 12,3 13,9
    Total volume 25 25 25


    Loading plan:

    M    P434  P437  P81  P195  P229
    


    Results of preparative agarose gel:
    Expected sizes:

      P434 = 3300bp
      P437 = 3183 bp
      P195 = 627bp, 2054bp
      P229 = 756bp, 667bp, 2600bp, 1300bp


    Freiburg10 Digestion of pSB1C3 CMV promoter bglob GOI.jpg


    After the gel extraction (the fragments cut out of the gel were labeled) has been performed the ligation was carried out.

    Ligation:

    • v=P434 =5,61µL
    • v=P195 =2,39µL


    • v=P437 =4,77µL
    • v=P195 =3,32µL


    • v=P437 =4,17µL
    • v=P229 =3,83µL

    After mixing insert and vector into a tube, add 1µL T4-ligase 10x buffer and 1µL ligase to the DNA mix and incubate for 40 minutes at room temperature.
    Transform XL1-B cells, and plate them on agar containing chloramphenicol.

    Next steps: Picking clones of the constructs, prepare Mini-prep and finally fuse them to the existing construct pSB1C3_hGH_rightITR!

    Sent the construct pSB1C3_VP2/3 and pSB1C3_VP2/3 for sequencing

    Investigator: Bea

    Comment: The construct which has been produced (experiment started on friday) will be sequenced today. The construct were named: P357 1- 3 and P396 1-3. After preparing for sequencing the plasmids were renamed and received a plasmid number aswell.

    • P357_1 = P440
    • P357_2 = P441
    • P357_3 = P442
    • P396_1 = P443
    • P396_3 = P443
    • P396_3 = P445


    The two constructs which have been sent for sequencing are:

    • P440 = BK_1, used primers: VR-2 and VF-2
    • P444 = BK_2, used primers: VR-2 and VF-2

    Fusion of VP2/3 to C-terminus of Zegfr:1907_"Linker", CFP_MiddleLinker and 6xHis_MiddleLinker construct

    Investigator: Hanna



    120. labday 14.09.2010

    Sequencing analysis from pSB1C3_VP2/3 and pSB1C3_VP2/3_ins_cap

    Investigator: Bea

    Comment: The constructs which were sent for sequencing are the plasmids which were PCR amplified from the pAAV_RC and cloned into the pSb1C3. Purpose of the PCR amplification was to clone the fragement to the N-terminal targeting approaches.

    • P440 = pSB1C3_VP2/3 = BK_1
    • Used primers:
      • VR-2
      • VF-2

    Results:

    Freiburg10 Seq analysis P440 BK1 VF2 13.09..jpg


    • P444 = pSB1C3_VP2/3_ins_cap = BK_2
    • Used primers:
      • VR-2
      • VF-2

    Results:


    Freiburg10 Seq analysis P444 BK2 VF2 13.09..jpg
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