Team:Freiburg Bioware/NoteBook/Labjournal/September

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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 145 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 146 - 155 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October3">October part 3 (labday 156 - 166 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture </a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
</ul>
</ul>
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</div>

Latest revision as of 21:34, 27 October 2010


=> Back to Notebook overview

Contents

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
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


    ;-) bitte ausfüllen

    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
    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

    Investigator: Kira
    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.

    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°C40"
    17x98°C15"
    65°C25"
    72°C40"
    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 CD 2010 09 07.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 klenow 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
    8x (step 2 - 4)9815
    6125
    723
    17x (step 5- 79815
    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 1
    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: The following primers were 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
    35x (step 2-4)9520
    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

    Investigator: 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

    Investigator: 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 2-4)9515
    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:
    0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, at 90 Volt, running time: ~90 minutes

    Freiburg10 pCerulean VP1up NLS targeting VP2 3.jpg


    Gel-Extraction:
    Marked bands on the gel were cut out. Gel extraction was performed according to protocol.

    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
    • Test Digestion ViralBricks:
    Comparison with control fragments shows that most clones dont contain a correct insert. (Insert sizes should be about 230 bp.) We sent sample 11.2 (Buffer 4) and 11.1 (Klenow buffer) for sequencing
    • Test Digestion Assembly
    2 distinct bands are visible, the expected sizes of 2000 and 2500 bp match with the ladder.

    PCR of RepCap/Cap

    Investigator: Jessica

    Dear Jessy, would you please always comment and explain what you're doing - so that other persons also have a chance to understand what you've done!

    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: O89 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


    Why did you use two times the same PCR program and the same primer for different approaches?

    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

    Freiburg10.jpg

    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

    UPDATE: The Zegfr:1907 Affibody was successfully fused to the different linker (Short Linker, Long Linker, Middle Linker and SEG Linker) in order to find out with which linker targeting functions best. In addition to that, CFP (Imaging) and the His-Tag (=6xHis, Purification) was fused to the Middle Linker. These constructs were cloned into the pCerulean plasmid (in order to be driven by the CMV promoter + SV40 terminator).
    The last steps in the VP2-N-terminal fusion approach include the knockout of the VP2 start codon in pAAV_RC (different constructs: 4-fold mutated, 4-fold mutated with Cap-insert, 4-fold mutated with Rep/Cap-insert/KpnI-backmutation) and fusion of the VP2/3 ORF to the C-terminus of the linker.

    SDM of the VP2 start codon (of the unmodified and Cap-insert sequence) was already performed by Stefan. After the Cap-insert variant has been tested in cell culture, the HSPG-KnockOut motif will be cloned into Cap and also used for BioBrick production via PCR. This fragment will also be used for fusion to the C-terminus of the Linker-Library+Affibody and the purification (6xHis) and Imaging (CFP, better: GFP) approaches. Therefore pAAV_RC_Rep/Capins_KpnI-backmutation_HSPG-KO (if Rep-KpnI-backmutation works!) will be also used for SDM of VP2 start codon.

    Today VP2/3 (unmodified) and VP2/3-Cap-ins will be fused to the C-terminus of the linker. These approaches will be co-transfected with the referring pAAV_RC_VP2-KO constructs and therefore tested in cell culture on wednesday.


    Practical Cloning:


      • Vector: pCerulean_CFP_MiddleLinker (P407), pCerulean_Zegfr:1907_MiddleLinker (P408), pCerulean_Zegfr:1907_SEG (P409), pCerulean_Zegfr:1907_ShortLinker (P410), pCerulean_Zegfr:1907_LongLinker (P412), pCerulean_6xHis_MiddleLinker (P374)
      • Insert: pSB1C3_VP2/3 (P440), pSB1C3_VP2/3_ins_cap (P444)
      • new vector name: pCerulean_CFP_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_SEG_VP2/3, pCerulean_Zegfr:1907_ShortLinker_VP2/3, pCerulean_Zegfr:1907_LongLinker_VP2/3, pCerulean_6xHis_MiddleLinker_VP2/3 AND pCerulean_CFP_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_SEG_VP2/3-insCap, pCerulean_Zegfr:1907_ShortLinker_VP2/3-insCap, pCerulean_Zegfr:1907_LongLinker_VP2/3-insCap, pCerulean_6xHis_MiddleLinker_VP2/3-insCap
    • buffer used:4

    Comment Because the VP2/3 frament has approximately the same size as the backbone pSB1C3 it may be difficult to separate them on the agarose gel. Therefore a single cutter enzyme (SacI) was chosen to digests the backbone. This enzyme also works in buffer 4 + BSA and needs to be incubated at 37°C.

      • Vector: AgeI-HF, SpeI
      • Insert: NgoMIV, SpeI, SacI



    Digestion

    components volume of vector /µl volume of insert /µl
    DNA 3.75 8.3
    BSA (10x) 2 3
    Buffer 4(10x) 2 3
    AgeI-HF 1 -
    NgoMIV - 1
    SpeI 1 1
    SacI - 1
    H2O 10.25 12.7
    Total volume 20 30
    • Incubation: 2 h

    Comment_ Because we are all hardworking cloners :) there was not enough AgeI left for all 6 reactions (just 3: P374, P407, P408). Unfortunately the lab stock AgeI was also empty. Therefor we had to "lend" it from the Baumeister group - which is already from "Fermentas" and therefore also needs Fermentas-buffer. Nevertheless we checked the pH from buffer 4 with the one needed for the Fermentas-AgeI and decided to try using this AgeI in buffer 4.

    Agarose-Gel:


    0.8 g and 0.4 g Agarose, 50 mL TAE (0.8 % and 1.5 %), 3 µL GELRED, at 90 Volt, running time:60 minutes Comment: Unfortunately the gels were interchanged so that the vector was loaded onto the 1.5% gel.

    Sample Sample/µl] Loading dye (6x)/µl Expected size 1 Expected size 2 Expected size 3
    P407 20 µl 4 µl ~ 4170 bp - -
    P408 20 µl 4 µl ~ 4170 bp - -
    P409 20 µl 4 µl ~ 4170 bp - -
    P410 20 µl 4 µl ~ 4170 bp - -
    P412 20 µl 4 µl ~ 4170 bp - -
    P374 20 µl 4 µl ~ 4170 bp - -
    P440 30 µl 6 µl 1900 bp 910 bp 1150 bp
    P444 30 µl 6 µl 1900 bp 910 bp 1150 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample Jessy /µl Sample P407 /µl Sample P408 /µl Sample P409 /µl Sample P410 /µl Sample P412 /µl
    Lane 5.5 24 24 24 24 24


    Sample P374 /µL Sample P440 /µl Sample P444 /µl Marker /µl
    Lane 24 36 36 5.5


    Freiburg10 VP23Fusion.png Freiburg10 VP23Fusion.2.png


    Gel extraction


    Gel measurement:

    Sample Weight
    P407 150 mg
    P408 180 mg
    P409 230 mg
    P410 220 mg
    P412 170 mg
    P374 180 mg
    P440 210 mg
    P444 180 mg




    Ligation


    Construct Vector (µl) Insert (µl)
    407 + 440 4.6 3.4
    407 + 440 4.98 3.02
    408 + 440 4.14 3.86
    408 + 444 4.53 3.47
    409 + 440 4.18 3.82
    409 + 444 4.57 3.43
    410 + 440 3.6 4.4
    410 + 444 3.99 4.01
    412 + 440 3.6 4.4
    412 + 444 3.99 4.01
    374 + 440 3.23 4.77
    374 + 444 3.61 4.39


    120. labday 14.09.2010

    Test Digestion of remaining ViralBrick clones

    Investigator: Achim

    None of the samples contained inserts of the expected size. (Either religated vector or BLA). Samples were discarded. New ligation tomorrow.

    ViralBrick sequencing results

    Investigator: Achim

    11.2, Buffer 4
    11.1, Klenowbuffer


    • The two plasmids contain Z34C, but are incomplete in the 453 upstream region. There could be several reasons for this: Incompletely synthesized oligos, incomplete fill-in reactions...
    • Tomorrow we're going to try another repetition of the hybridisation and ligation, this time with dephosohorized vector - that should prevent the growth of clones containing religated vector. We will also use new Klenow fragment and try two parallel ligations: Quickligation & T4 ligation over night.

    Retrafo with sr39_rightITR (Mr.Gene)

    Investigator: Bea

    Comments: We received the sr39 (mutant verstion of the wt thymidinkinase) and wt rightITR ordered from Mr. Gene today. In order to use and clone the insert into the desired sequences a retrafo has to be performed.

    Re-transformation:

    • Construct used:
    • We reveived 5 µg (lyophilzied). These were resuspended in 100 µL H2O and vortexted.
    • c = 50 ng/µL
    • c = Plasmid need to be diluted in order to obatain the final 500pg
    • Dilution: 1:100
    • 100 µL aliquot of XL1-B were thawed on ice. The proper amount of 1µL was added to the cells.
    • Transformed cells were plated on LB plates containing kanamycin

    Picking clones of pSB1C3_leftITR_promoter_betaglobin_GOI

    Investigator: Bea

    Comments: Clones were picked from the approach condutced yesterday.

    • pSB1C3_leftITR_CMV_betaglobin_mGMK
    • pSB1C3_leftITR_phTERT_betaglobin_mGMK
    • pSB1C3_leftITR_phTERT_betaglobin_mVenus

    Additionally, pHelper (Glycerol stock) was inoculated in 30 mL containing ampicillin.

    3rdrepetition of SV40 terminator (hopefully the last one ;-))

    Investigator: Bea

    Comments: The last repetition of the PCR for obtaining the SV40 fragment did not work out because wrong primers were used therefore the PCR will be carried out once again.


    Preparations:
    A 1:1000 dilution of the plasmid P230 (pEGFP_C1) which contains the desired SV40 polyadenlyation site will be prepared. This fragment will be extracted and cloned into pSB1C3 iGEM standard plasmid.

    PCR protocol:

    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 Template3,7 µl
    DMSO 0 µl
    Phusion Polymerase0,5 µl
    H2O29,8 µl
    Total volume50 µl


    The following PCR programm was used. Idea is that in the first 8 cycles the primer can only bind to the template added in to the reaction mix. The following 17 cycles afterwards allow the annealing of the complete primer containing prefix and suffix with its restriciton sites.

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


    The PCR product (after PCR purification) and the plasmid vector pSB1C3_CFP (P51.2) will be digested with the same restriction enzymes and afterwards loaded on a 1% agarose gel:

    • XbaI
    • PstI
    Components vPCR product SV40 /µL vP51.2 /µL
    DNA 209
    BSA (100x) 0,3 0,2
    Buffer no. 4 (10x) 3 2
    SpeI 1 1
    PstI-HF 1 1
    H2O 4,7 6,8
    Total volume 30 20


    After digestion of the two constructs they were loaded on a 1% agarose gel.

    M    PCR product SV40    pSB1C3_CFP
    

    Expected sizes of the two digested constructs are:

    • PCR product SV40 terminator: 255bp
    • pSB1C3: 2053bp
    • pSB1C3: 756bp


    Results:

    PCR fragment was cut out after 20 minutes running the gel. The vector was cut out after 40 minutes.


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

    Ligation:

    • v=pSB1C3 =7,7µL
    • v=SV40 =0,3µL

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

    Next steps:
    Picking clones and perform Mini-Prep. This construct can then be submitted to the parts registry after sequencing.

    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
    ´

    Midiprep of pAAV_RC_CapIns_NoRep; pAAV_RC_RepCapIns_SDMKpnI

    Investigator: Chris W.
    The Midiprep was performed according to the standard protocol yieldung the following concentrations:

    pAAV_RC_CapIns_NoRep: 387,05 ng/µl, labeled as P467
    pAAV_RC_RepCapIns_SDMKpnI: 1157.58 ng/µl labeled as P468

    Sequencing analysis from pAAV_RC_RepCapIns_SDMKpnI

    Investigator: Stefan

    Comment: Last week's SDM to remutate the KpnI restriction site in Rep needed to be sequenced.

    • P432 = pAAV_RC_RepCapIns_SDMKpnI = AM_3
    • Used primers: 1250 for

    Results:

    Freiburg10 Sequencing pAAV RC RepCapIns SDMKpnI.jpg

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

    Investigator: Anissa

    COMMENT: The same approach which has been done yesterday, was repeated today, because gel-pictures showed no satisfying results.


    Practical Cloning:


      • Vector: pCerulean_CFP_MiddleLinker (P407), pCerulean_Zegfr:1907_MiddleLinker (P408), pCerulean_Zegfr:1907_SEG (P409), pCerulean_Zegfr:1907_ShortLinker (P410), pCerulean_Zegfr:1907_LongLinker (P412), pCerulean_6xHis_MiddleLinker (P374)
      • Insert: pSB1C3_VP2/3 (P440), pSB1C3_VP2/3_ins_cap (P444)
      • new vector name: pCerulean_CFP_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_SEG_VP2/3, pCerulean_Zegfr:1907_ShortLinker_VP2/3, pCerulean_Zegfr:1907_LongLinker_VP2/3, pCerulean_6xHis_MiddleLinker_VP2/3 AND pCerulean_CFP_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_SEG_VP2/3-insCap, pCerulean_Zegfr:1907_ShortLinker_VP2/3-insCap, pCerulean_Zegfr:1907_LongLinker_VP2/3-insCap, pCerulean_6xHis_MiddleLinker_VP2/3-insCap
    • buffer used:4

    Comment Because the VP2/3 frament has approximately the same size as the backbone pSB1C3 it may be difficult to separate them on the agarose gel. Therefore a 0,8% gel was prepared and the samples run for 1,5 h. So the two bands (1900bp and 2200bp) will hopefully be seperated.

      • Vector: AgeI-HF, SpeI
      • Insert: NgoMIV, SpeI



    Digestion

    components pSb1C3_Vp2/3 (2,5µg) P440 /µl /µl pSb1C3_Vp2/3_capIns (2,5µg) P444 /µl
    DNA 8,6 8
    BSA (10x) 1,5 1,5
    Buffer 4(10x) 1,5 1,5
    speI 1 1
    NgoMIV 1 1
    SpeI 1 1
    H2O 1,4 2
    Total volume 15 15


    components pCerulean_affib._shortlinker P410 (1µg)/µl pCerulean_affib._middlelinker P408 (1µg)/µl
    DNA 2 2,5
    BSA (10x) 1,5 1,5
    Buffer 4(10x) 1,5 1,5
    speI 1 1
    AgeI 0,75 0,75
    SpeI 1 1
    H2O 8,25 7,75
    Total volume 15 15


    components pCerulean_affib._longlinker P412 (1µg)/µl pCerulean_affib._SEG P409 (1µg)/µl
    DNA 2,4 2,4
    BSA (10x) 1,5 1,5
    Buffer 4(10x) 1,5 1,5
    speI 1 1
    AgeI 0,75 0,75
    SpeI 1 1
    H2O 7,85 7,85
    Total volume 15 15


    components pCerulean_CFP_middlelinker P407 (1µg)/µl pCerulean_His-tag._middlelinker P374 (1µg)/µl
    DNA 2,1 2,5
    BSA (10x) 1,5 1,5
    Buffer 4(10x) 1,5 1,5
    speI 1 1
    AgeI 0,75 0,75
    SpeI 1 1
    H2O 8,15 7,84
    Total volume 15 15
    • Incubation: 1,5 h

    Comment: 0,75 µl AgeI were used because AgeI is not deluted.

    Agarose-Gel:


    0.4 g and 0,5g Agarose, 50 mL TAE (0.8 %, 1%), 3 µL GELRED, at 110 Volt, running time:1,5 h
    


    Sample Sample/µl] Loading dye (6x)/µl Expected size 1 Expected size 2 Expected size 3
    P407 20 µl 4 µl ~ 4170 bp - -
    P408 20 µl 4 µl ~ 4170 bp - -
    P409 20 µl 4 µl ~ 4170 bp - -
    P410 20 µl 4 µl ~ 4170 bp - -
    P412 20 µl 4 µl ~ 4170 bp - -
    P374 20 µl 4 µl ~ 4170 bp - -
    P440 30 µl 6 µl 1900 bp 910 bp 1150 bp
    P444 30 µl 6 µl 1900 bp 910 bp 1150 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample p410 /µl Sample P408 /µl Sample P412 /µl Sample P409 /µl Sample P407 /µl Sample P374 /µl
    Lane 3 19 19 19 19 19 19


    Marker /µl Sample P444 /µl Sample P440 /µl


    Lane 3 19 19



    Gel extraction


    Gel measurement:

    Sample Weight
    P407 180 mg
    P408 160 mg
    P409 90 mg
    P410 190 mg
    P412 230 mg
    P374 190 mg
    P440 120 mg
    P444 130 mg




    Ligation


    Construct Vector (µl) Insert (µl)
    407 + 440 1 3
    407 + 440 1 3
    408 + 440 1 3
    408 + 444 1 3
    409 + 440 1 3
    409 + 444 1 3
    410 + 440 1 3
    410 + 444 1 3
    412 + 440 1 3
    412 + 444 1 3
    374 + 440 1 3
    374 + 444 1 3


    Comment: Because the amount of the insert-DNA wouldn't sufficiend for all ligations, the amount of the totl DNA was reduced to 4 µl.

    Transformation


    Transformation was performed in Xl1 blue according the standard-protocol.

    Mini-Prep and test digestion of multiple plasmids

    Investigator: Stefan

    Glycerol stocks were prepared:

    • B382 = pAAV_RC_1.2SDMSalI_VP1-ko clone 1
    • B383 = pAAV_RC_1.2SDMSalI_VP1-ko clone 2
    • B384 = pAAV_RC_CapIns_prepSDM_VP1-ko clone 1
    • B385 = pAAV_RC_CapIns_prepSDM_VP1-ko clone 2
    • B386 = pAAV_RC_CapIns_noRep_VP1-ko clone 1
    • B387 = pAAV_RC_CapIns_noRep_VP1-ko clone 2
    • B388 = pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 1
    • B389 = pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 2
    • B390 = pAAV_RC_1.2SDMSalI_VP2-ko clone 1
    • B391 = pAAV_RC_1.2SDMSalI_VP2-ko clone 2
    • B392 = pAAV_RC_CapIns_prepSDM_VP2-ko clone 1
    • B393 = pAAV_RC_CapIns_prepSDM_VP2-ko clone 2
    • B394 = pAAV_RC_CapIns_noRep_VP2-ko clone 1
    • B395 = pAAV_RC_CapIns_noRep_VP2-ko clone 2
    • B396 = pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 1
    • B397 = pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 2


    • B398 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3 clone 1
    • B399 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3 clone 2
    • B400 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3-Capins clone 1
    • B401 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3-Capins clone 2
    • B402 = pCerulean_VP1up_NLS_6xHis_VP2/3 clone 1
    • B403 = pCerulean_VP1up_NLS_6xHis_VP2/3 clone 2
    • B404 = pCerulean_VP1up_NLS_6xHis_VP2/3-Capins clone 1
    • B405 = pCerulean_VP1up_NLS_6xHis_VP2/3-Capins clone 2
    • B406 = pCerulean_VP1up_NLS_mVenus_VP2/3 clone 1
    • B407 = pCerulean_VP1up_NLS_mVenus_VP2/3 clone 2
    • B408 = pCerulean_VP1up_NLS_mVenus_VP2/3-Capins clone 1
    • B409 = pCerulean_VP1up_NLS_mVenus_VP2/3-Capins clone 2



    Mini-Prep was performed according to the standard protocol

    • P449 = pAAV_RC_1.2SDMSalI_VP1-ko clone 1 c= 372,90 ng/µl
    • P450 = pAAV_RC_1.2SDMSalI_VP1-ko clone 2 c= 380,47 ng/µl
    • P451 = pAAV_RC_CapIns_prepSDM_VP1-ko clone 1 c= 367,08 ng/µl
    • P452 = pAAV_RC_CapIns_prepSDM_VP1-ko clone 2 c= 387,34 ng/µl
    • P453 = pAAV_RC_CapIns_noRep_VP1-ko clone 1 c= 360,67 ng/µl
    • P454 = pAAV_RC_CapIns_noRep_VP1-ko clone 2 c= 353,62 ng/µl
    • P455 = pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 1 c= 353,85 ng/µl
    • P456 = pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 2 c= 349,39 ng/µl
    • P457 = pAAV_RC_1.2SDMSalI_VP2-ko clone 1 c= 308,46 ng/µl
    • P458 = pAAV_RC_1.2SDMSalI_VP2-ko clone 2 c= 342,85 ng/µl
    • P459 = pAAV_RC_CapIns_prepSDM_VP2-ko clone 1 c= 386,43 ng/µl
    • P460 = pAAV_RC_CapIns_prepSDM_VP2-ko clone 2 c= 327,22 ng/µl
    • P461 = pAAV_RC_CapIns_noRep_VP2-ko clone 1 c= 373,94 ng/µl
    • P462 = pAAV_RC_CapIns_noRep_VP2-ko clone 2 c= 347,39 ng/µl
    • P463 = pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 1 c= 372,55 ng/µl
    • P464 = pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 2 c= 348,33 ng/µl


    • P465 = pSB1C3_SEG c= 208,32 ng/µl
    • P466 = pSB1C3_mGMK c= 224,62 ng/µl


    • P470 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3 clone 1 c= 53,04 ng/µl
    • P471 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3 clone 2 c= 114,24 ng/µl
    • P472 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3-Capins clone 1 c= 170,16 ng/µl
    • P473 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3-Capins clone 2 c= 145,98 ng/µl
    • P474 = pCerulean_VP1up_NLS_6xHis_VP2/3 clone 1 c= 104,29 ng/µl
    • P475 = pCerulean_VP1up_NLS_6xHis_VP2/3 clone 2 c= 147,86 ng/µl
    • P476 = pCerulean_VP1up_NLS_6xHis_VP2/3-Capins clone 1 c= 97,33 ng/µl
    • P477 = pCerulean_VP1up_NLS_6xHis_VP2/3-Capins clone 2 c= 97,94 ng/µl
    • P478 = pCerulean_VP1up_NLS_mVenus_VP2/3 clone 1 c= 133,61 ng/µl
    • P479 = pCerulean_VP1up_NLS_mVenus_VP2/3 clone 2 c= 107,60 ng/µl
    • P480 = pCerulean_VP1up_NLS_mVenus_VP2/3-Capins clone 1 c= 45,80 ng/µl
    • P481 = pCerulean_VP1up_NLS_mVenus_VP2/3-Capins clone 2 c= 115,16 ng/µl



    Test digestion:
    No test digestion was performed for Plasmid numbers P449 to P466. P449 to P464 were obtained from a SDM to knock-out VP1 or VP2. There is no possibility to verify this from a test digestion. P465 and P466 were obtained from glycerol stock and therefore no test digestion is necessary. The other constructs were digested using EcoRI and SpeI for 40 minutes at 37°C.


    components P470-P481
    DNA 2
    Buffer 1 (10x)1
    BSA (10x)1
    EcoRI 0,5
    SpeI 0,5
    H2O5
    Total volume 10


    Preparation of gel:

    • 0,4 g Agarose, 50 ml TAE (0,8%), 3 µl GELRED , at 110 Volt, running time: 45 minutes


    Freiburg10 test digestion pCerulean VP1up NLS targeting VP2 3.jpg

    Comment: it is still not clear whether or not the correct insert was cloned into pCerulean. Therefore, P475 and P473 will be sent for sequencing tomorrow using pEGFP_C2-RP as primer to verify VP2/3 insertion.

    Sent for sequencing:
    Primer used:
    2800 reverse (O38)

    • P449
    • P451
    • P453
    • P455
    • P457
    • P459
    • P461
    • P463

    121. labday 15.09.2010

    New ViralBrick cloning attempt

    Investigator: Achim

    Yet another attempt at hybridizing and ligating the ViralBrick oligos into the pSB1C3_BLA. Changes:

    • Dephosphorylation of digested vector to prevent religation
    • New Klenow Fragment from Fermentas
    • Two parallel ligation approaches:
      • Quickligation for 10 min
      • T4 Ligation over night

    Following steps were carried out:

    • Hybridisatzion of oligos
      • 11:453 Z34C: 143 & 144
      • 12:587 Z34C: 145 & 146
      • 13:587 KO Z34C: 147 & 148
      • 14:587 KO Z34C: 149 & 150

    Condtitions as before:

    • 2 µg DNA, (3,3 µl of each oligo)
    • 2 µl Klenow buffer
    • 0,1 µl dNTPs (1µl of 1:10 dilution of our aliquots)
    • 9,8 µl H2O

    Total volume: 20 µl

    Hybridisation was carried out on the gradient cycler using the program "tkfill"

    0,5 µl Klenow Fragment was added after the temperature reached 37°C. Klenow reaction was carried out for 1 h in the 37°C room.

    Afterwards, the samples were purified with the Quiagen PCR Purification kit. Elution in 50 µl H2O. Nanodrop concentrations:

    • 11: 47.08 ng/µl
    • 12: 28.50 ng/µl
    • 13: 37.69 ng/µl
    • 14: 42.98 ng/µl

    1 µl of SspI & SalI for sample 11/ BamHI & PvuII for samples 12,13,14 as well as 5,8 µl buffer 4 for each sample were added to the purified samples. Digestion for 3 h at 37 °C

    The vector backbone pSB1C3_VCK_BLA (P320)was also digested with BamHI/PvuII and SspI/SalI for 3 h. Afterwards,the cut vector fragment was dephosphorylated with Antarctic Phosphatase for 1h at 37°C.

    The oligos and the vector fragments were mixed with loading dye and run over a preparative gel (1%):

    Oligos ran between 100 - 200 bp, the unhybridized oligo control ran a bit further.

    Fragments were cut out, followed by gel extraction. Two ligations were prepared: T4-Ligation and Quickligation.

    Quickligation: 10 min, followed by trafo. Plates were incubated overnight.

    T4-Ligation: Overnight

    Cloning VP2/3 approaches into pSB1C3_VCK vector

    Investigator: Bea

    Comment:Since the constructs VP2/3 and VP2/3_ins_cap were cloned in the plasmid pSB1C3 they need to recloned into the vector pSB1C3_VCK which have two deleted restrictions sites (PvuII ans SspI) . These restriction sites have to be single cutting enzymes in this case because we want to subclone the HSPG-knockout "motif" into the cap gene in order to obtain constructs for the N-terminal targeting approach. The recombinant virus particles basically then do not show the natural tropism for its primary receptor.


    Digestion of the constructs:

    • P440 = pSB1C3_VP2/3 c=290 ng/µL
    • P440 = pSB1C3_VP2/3_ins_cap c=314ng/µL
    • P309 = pSB1C3_VCK_BLA c=408ng/µL


    Components vP440 /µL vP444/µL vP309 /µL
    DNA 5,5 5,52,5
    BSA (10x) 4 4 3
    Buffer no. 4 (10x) 4 4 3
    EcoRI 1 1 1
    AgeI 1 1 1
    MISC SacI: 1 MscI: 1 -
    H2O 23,5 23,5 19,5
    Total volume 40 40 30


    The expected fragments of the digested constructs were: The underlined fragments represent the fragments which correspond to the desired construct which can be used for ligation.

    • P440: 1950, 1158, 908bp
    • P444: 1950, 1347, 715bp
    • P309: 2067, 894bp


    Loading plan:

    M    P440    P444    P309
    


    Results:

    Freiburg10 pSB1C3 VCK VP2 3 15 09 2010.jpg


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

    Ligation:

    • v=P444 =5,5µL
    • v=P309 =2,5µL

    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 these constructs can be used for inserting the HSPG knockout motif.

    Colony PCR of CD colonies

    Investigator: Kira

    Comments: Since the last approach with the CD did not work, we performed a colony PCR in order to detect the positive clones which should contain the CD gene. We used the two SDM primers, originally used for deleting the iGEM restriction sites in the CD gene, which should only bind to the CD sequence.


    15 colonies were picked from the plate and dispersed first into the PCR tube and then into DYT media as a backup in case of successful PCR reaction.

    components pro sample
    10x Taq Buffer 1
    MgCl (50mM) 0,1
    dNTPs0,2
    Primer 1 0,2
    Primer 2 0,2
    Taq Polymerase 0,05
    H2O8,25
    Total volume 10


    PCR program:

    CyclesTemperatureTime
    95°C6 min
    30x95°C25 sec
    63°C30 sec
    68°C1 min 6 sec
    1x68°C5 min
    Hold 4°C


    pSB1C3_RFC25_CFP was used as a control sample

    1% agarose gel

    Freiburg10 colony pcr 2010 09 15.jpg

    According to the gel, clone 3, 6, 7, 9, 11, 14 contain CD gene. These samples will be incubated over-night @ 37C.


    Comment added on 17.09.2010 : We (Bea, Kira, Stefan and Tobi) wondered why sequencing of one of the positive clones of the colony PCR revealed that the construct did not contain the expected CD gene but CFP instead. Today, we realized that the colony PCR does not reveal only positive constructs which contain the cytosine deaminase. Problem is that we received the CD gene originating from the COD-BA gene of E. coli. Since we performed a colony PCR with XL1-B E. coli cells the used primers will bind to the genomic CD-gene aswell which could explain the positive clones in the gel, the negative results of seuqencing and another test digest of hte sequenced clone.
    Next steps are that we are performing the colony PCR again with new primers which means that we are using the iGEM primers which should specifically bind to the constructs which contain the iGEM prefix and suffix add-ons. Since CFP does contain the iGEM restriction sites aswell, but differ in the size of the expected fragment (CD ~1100bp vs. CFP ~700bp) the positive clones should be detectable by the size length.


    Cloning of pSB1C3_Affibody_VP2/3

    Investigator: Jessica

    Comment: Cloning of Affibody and VP2/3 without linker to test if it works without linker


    Digestion:

    components pSB1C3_ZEGFR:1907 clone 2 (P268) pSB1C3_VP2/3 clone 1 (P440)
    DNA 14,35 5,2
    BSA 2 2
    Buffer 4 (10x)2 2
    XbaI 1 1
    NgoMIV -1
    AgeI 1-
    H2O- 8,8
    Total volume 20,35 20


    Comment:Digestion was performed 1,5 hours at 37 °C



    Gel:

    0,5 g Agarose,50 ml TAE (1%), 5 µl Gelred , at Volt
    Freiburg10 gelex affibody vp23.jpg

    Gelextraction:

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

    • pSB1C3_ZEGFR:1907 clone 2 (P268): c= 2,6ng/µl
    • pSB1C3_VP2/3 clone 1 (P440: c= 6,7ng/µl


    Ligation:

    The Ligation was performed as following:

    • Vector Volume: 5,83µl
    • Insert Volume: 2,17µl


    • 1 µl T4Ligase buffer (2x)
    • 8 µl (Vector + Insert) mix
    • 1 µl T4Ligase


    Incubating for 45 minutes.


    Transformation:

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

    PCR of GFP_RFC25 and cloning into pSB1C3_6XHis and pSB1C3_CFP

    Investigator: Anissa

    Comment:GFP will be cloned into pSB1C3 for biobrick production and upstream of his-tag into into pSB1C3_his for further use


    Preparations:
    A 1:1000 dilution of the plasmid P416 (pSB1A3_EGFP_HQ clone1). GFP will be extracted and cloned into pSB1C3 iGEM standard plasmid with the RFC25 standard.

    PCR protocol:

    Ingredients volume
    5X Phusion HF buffer 10 µl
    10 mM dNTP mix1µl
    forward primer: O186 2,5 µl
    reverse primer: O187 2,5 µl
    DNA Template5 µl
    DMSO 0 µl
    Phusion Polymerase0,5 µl
    H2O28,5 µl
    Total volume50 µl


    The following PCR programm was used. Idea is that in the first 8 cycles the primer can only bind to the template added in to the reaction mix. The following 17 cycles afterwards allow the annealing of the complete primer containing prefix and suffix with its restriciton sites.

    CyclesTemperatureTime
    98°C1
    8x98°C15"
    54°C25"
    72°C24"
    17x98°C15"
    64°C25"
    72°C7"
    1x72°C5'
    Hold 4°C


    Digestion of the vectors:

    components pSB1C3_CFP (P51.2) pSB1C3_6XHis (P300)
    DNA 6,6 5,6
    BSA 1,5 1,5
    Buffer 4 (10x)1,5 1,5
    XbaI 1 1
    NgoMIV -1
    AgeI 1-
    H2O3,4 4,4
    Total volume 15 15


    Comment:Digestion was performed 1,5 hours at 37 °C



    Gel:

    0,5 g Agarose,50 ml TAE (1%), 3 µl Gelred , at 110 Volt

    Comment:The gel showed for the sample p300 no clear band. Perhaps the enzymes did not cut right. The gel-ex-sample of p300 was seperated into an "upper" and a "lower band" sample.




    Gelextraction:

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

    • pSB1C3 (P51.2): c= 3,95 ng/µl
    • pSB1C3_6Xhis_upper (P300): c= 8,46 ng/µl
    • pSB1C3_6Xhis_lower (P300): c= 2,10 ng/µl
    • PCR GFP_RFC25: c= 6 ng/µl



    Digestion of the PCR-product:

    components GFP_RFC25
    DNA 30
    BSA 4
    Buffer 4 (10x)4
    XbaI 1
    AgeI 1
    H2O-
    Total volume 40


    Comment:Digestion was performed 1,5 hours at 37 °C



    Purification of the PCR-product: Was performed accoding to the standard-protocol


    Ligation:

    The Ligation was performed as following:

    • P51.2 Volume: 5,83µl
    • Insert Volume: 2,17µl


    • P300_upper Volume: 5,83µl
    • Insert Volume: 2,17µl


    • P300_lower Volume: 5,83µl
    • Insert Volume: 2,17µl


    • 1 µl T4Ligase buffer (2x)
    • 8 µl (Vector + Insert) mix
    • 1 µl T4Ligase


    Incubating for 30 minutes.


    Transformation:

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

    PCR for biobrick production: Cloning RepCap into pSB1C3

    Investigator: Stefan

    Comment:Goal of the experiment was to bring the different RepCap constructs into the pSB1C3 plasmid containing the deletions of the restriction sites interfering with the loop insertions.


    Preparations:
    Plasmids used:

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

    A 1:1000 dilution of each of the plasmids was prepared.

    PCR protocol:

    Ingredients P158 (v/µl) P396 (v/µl) P413 (v/µl) P432 (v/µl)
    5X Phusion HF buffer 10 10 10 10
    10 mM dNTP mix1111
    forward primer: O93 2,5 2,5 2,5 2,5
    reverse primer: O120 2,5 2,5 2,5 2,5
    DNA Template2,523,23
    DMSO ----
    Phusion Polymerase0,50,50,50,5
    H2O3131,530,330,5
    Total volume50505050


    PCR program:

    Comment:For all approaches the following programm was used:


    CyclesTemperature /°CTime /s
    19860
    2 (8x step 2-4)9815
    35925
    47270
    5 (17x step 5-6)9815
    67280
    71x72300
    Hold 4°C


    Digestion of the vector:

    components pSB1C3_VCK_Bla (P320) (v/µl)
    DNA 6,1
    BSA 2
    Buffer 4 (10x)2
    XbaI 1
    AgeI 1
    H2O7,9
    Total volume 20


    Comment:Digestion was performed 2 hours at 37 °C.



    Gels:
    For PCR products:
    0,4 g Agarose,50 ml TAE (0,8%), 3 µl Gelred , at 110 Volt; run for ~40 minutes

    Freiburg10 Biobrick PCR of RepCap.jpg

    For vector:
    0,5 g Agarose,50 ml TAE (1%), 3 µl Gelred , at 110 Volt; run for ~50 minutes

    Freiburg10 Biobrick vector digest of RepCap.jpg



    Gelextraction:

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

    • pSB1C3 (P320): c= 11,97 ng/µl



    Digestion of the PCR-products:

    Comment:Digestion was performed for all PCR products alike.


    components PCR products (v/µl)
    DNA 30
    BSA 4
    Buffer 4 (10x)4
    XbaI 1
    AgeI 1
    H2O-
    Total volume 40


    Comment:Digestion was performed 2 hours at 37 °C.



    PCR purification:
    Purification was performed accoding to the standard protocol. The following concentrations were measured:

    • PCR of P158: c= 7,68 ng/µl
    • PCR of P396: c= 17,65 ng/µl
    • PCR of P413: c= 24,17 ng/µl
    • PCR of P432: c= 18,23 ng/µl


    Ligation:
    1 µl T4 Ligase buffer (2x) 8 µl DNA mix 1 µl T4 Ligase
    Incubating for 45 minutes at room temperature.

    The Ligation was performed using the following amounts of DNA:

    • Vector Volume: 1,98 µl
    • P158 Volume: 6,02 µl

    • Vector Volume: 3,45 µl
    • P396 Volume: 4,55 µl

    • Vector Volume: 4,07 µl
    • P413 Volume: 3,93 µl

    • Vector Volume: 3,51 µl
    • P413 Volume: 4,49 µl



    Transformation:

    Trafo was performed according to the standard protocol using XL1b cells. The cells were plated on a agar plates containing chlorampenicol.

    Mini-Preps

    Investigator: Hanna

    Mini-Preps were performed according to standard protocol:
    pSB1C3_ITR_phTERT_b-globin__mGMK clone 1 (P483)
    pSB1C3_ITR_phTERT_b-globin__mGMK clone 2 (P484)
    pSB1C3_lITR_phTERT_b-globin__mVenus clone 1 (P485)
    pSB1C3_lITR_phTERT_b-globin__mVenus clone 2 (P486)
    pSB1C3_lITR_CMV_b-globin__mGMK clone 1 (P487)
    pSB1C3_lITR_CMV_b-globin__mGMK clone 2 (P488)
    pSB1C3_VP123 (aus pAAV_RC_CapIns_noRep P413) clone 1 (P489)
    pSB1C3_VP123 (aus pAAV_RC_CapIns_noRep P413) clone 2 (P490)
    pSB1C3_VP123 (aus pAAV_RC_1.2 SDM SalI P158) clone 1 (P491)
    pSB1C3_VP123 aus (pAAV_RC_1.2 SDM SalI P158) clone 2 (P492)

    Colony PCR of pCerulean_CFP_MiddleLinker, pCerulean_6xHis_MiddleLinker and pCerulean_"Linker"_Zegfr:1907 constructs (Linker library)

    Investigator: Hanna

    Comment: Because we found out that there's also a SacI restriction site in the VP2/3 sequence - due to the PstI (4073) mutagenesis - the first try of cloning VP2/3 to the C-terminus of the Zegfr:1907, CFP and His-Tag constructs was discarded.
    Fortunately Anissa already repeated this experiment yesterday. Because we're not sure whether cloning was successful (one plate was without any colonies and has to be done one more time), we decided to perform a colony PCR before continuation with (at least 22!) mini-preps. We chose primer which bind in the VP2/3 region and therefore amplify a VP2/3 fragment - given, that VP2/3 was successfully cloned downstream of the linker. One further advantage is, that we can also save the test digestions.
    A Mastermix was prepared for 43 samples:

    • 5x Crimson Taq Mg-free reaction buffer: 215 µL
    • 10 mM dNTP: 21.5 µL
    • 10 µM Forward primer (Primer Cap 3500 for): 21.5 µL
    • 10 µM Reverse Primer (Primer Cap 4200 rev): 21.5 µL
    • Crimson Taq DNA Polymerase: 5.375 L
    • Nuclease-Free water: 790.125 µL

    Total: 1075 µL -> 25 µL reactions

    2-4 colonies were picked with a tooth pick from each plate (11). 25 µL master mix and 5 ml LB + Kanamycin were inocculated.

    PCR program:
    According to standard protocol provided by Gerrit

    • 95°C, 30
    • 95°C 25
    • 50.5°C, 40
    • 68°C, 60 --> repeat 2-4 27 times
    • 68°C, 300
    • 4°C, hold



    Tomorrow morning the PCR products will be tested on an analytic gel. If the samples will be positive, they will be mini-preped - if not cloning will be done again.
    Tod do: Cloning of VP2/3 into pCerulean_CFP_MiddleLinker.

    Sequencing analysis of VP1-ko and VP2-ko in severeal pAAV vectors

    Investigator: Stefan
    Primer used (for all plasmids): 2800 rev (O38)

    Sequenced plasmid:

    • pAAV_RC_1.2SDMSalI_VP1-ko clone 1 (P449)
    • pAAV_RC_CapIns_prepSDM_VP1-koclone 1 (P451)
    • pAAV_RC_CapIns_noRep_VP1-ko clone 1 (P453)
    • pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 1 (P455)
    • pAAV_RC_1.2SDMSalI_VP2-ko clone 1 (P457)
    • pAAV_RC_CapIns_prepSDM_VP2-ko clone 1 (P459)
    • pAAV_RC_CapIns_noRep_VP2-ko clone 1 (P461)
    • pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 1 (P463)

    Comment:All sequences showed that the site directed mutagenesis worked out for all constructs. All sequencing results revealed problems in the area shown below:

    Freiburg10 Sequencing VP1 2 ko problem(2).jpg


    Freiburg10 Sequencing VP1 2 ko problem(1).jpg

    122. labday 16.09.2010

    Trafo of T4 ligation, Results of Quickligation

    Investigator: Achim


    No clones on the Quickligation plates. Trafo of T4 Ligation was carried out in XL1B cells. Results will be evaluated tomorrow.

    Mini Prep and test digestion of several constructs

    Investigator: Anissa, Bea

    Comment: Several test digestions have been performed for verifying the cloned fragment into the plasmid backbone. The constructs for reassembling the vector plasmid (pSB1C3_leftITR_PROMOTER_betaglobin_GOI) were digested with EcoRI and NgomIV, the SV40 construct was digested with EcoRI and PstI.


    Test digestion of the constructs:

    • pSB1C3_leftITR_phTERT_betaglobin_mVenus (P485-486)
    • pSB1C3_leftITR_phTERT_betaglobin_mGMK (P483-484)
    • pSB1C3_leftITR_pCMV_betaglobin_mGMK (P487-488)
    • pSB1C3_SV40 (P493-494)


    Expected size fragments are:

    • (P485-486): 1150bp, 2780bp
    • (P483-484): 1150bp, 2650bp
    • (P487-488): 1300bp, 2600bp
    • (P493-494): 270bp, 2030bp


    Results: Test digestion of the constructs looks well, except for the construct 494 which is one clone of the construct pSB1C3_SV40. Since the other clone is fine, the construct was discarded. The control K437 is the plasmid pSB1C3_leftITR_hTERT_beta-globin without the assembled GOI (mGMK and mVenus repsectively). The GOI´s are in the RFC25 standard therefore leading to scar with a additional NgoMIV after fusing it to the before mentioned construct which is in the RFC10 standard. Therefore, test digestion was carried out with NgoMIV and EcoRI leading to fragments which can be separated from the backbone in a 1% agarose gel. The control vector should only be linearized because it does not contain any NgomIV rstriction site.

    Freiburg10 pSB1C3 leftITR Promoter betaglob GOI 16 09 2010.jpg

    Test digestion of pSB1C3_VP123

    Investigator: Jessica


    Samples Plasmid Clone Concentration [ng/µl]
    P489  pSB1C3_VP123 (aus pAAV_RC_CapIns_noRep P413) 1 350
    P490  pSB1C3_VP123 (aus pAAV_RC_CapIns_noRep P413) 2395,5
    P491 pSB1C3_VP123 (aus pAAV_RC_1.2 SDM SalI P158) 1403
    P492 pSB1C3_VP123 (aus pAAV_RC_1.2 SDM SalI P158)2411


    Test digestion:

    components Volume for each sample /µl
    DNA 2,6/2,3/2,3/2,2
    BSA (10x) 1
    Buffer 4 (10x) 1
    Enzyme NgoMIV 0,5
    Enzyme SapI 0,5
    H2O 4,4/4,7/4,7/4,8
    Total volume /µl 10


    Preparation of gel:

    • Incubation time: 1,5 h, Incubation temperature: 37°
    • 0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at Volt, running time: minutes



    Freiburg10 Test digestion VP123.jpg

    • Expected fragment sizes:
    3244 bp
    767 bp

    Comment: Sample P489 was sent for sequencing with primer VR2. Sequencing result looks good, VP123 is in

    Continuation of preparation of competent E.coli

    Investigator: Jessica
    preparation of competent BL21 was finished according to the standard protocol

    • aliquots of 60µl (to use for 1 trafo) are stored in -80°C freezer

    don't use before results of testtrafo is writen down
    testtrafo was made with pUC and Amp


    Mini prep of CD-clones, which were identified by colony pcr

    Investigator: Kira

    c(clone1) = 160,29 ng/ul
    c(clone2) = 167,98 ng/ul
    c(cloone3) = 181, 51 ng/ul

    clone 3 was sent for sequencing

    Repetition: PCR for biobrick production of P40 promotor

    Investigator: Stefan

    Comment: During trouble-shooting we realized that the reverse primer P40_rev (O153) was designed forward and therefore does not work. A new primer will have to be designed and ordered to repeat the PCR.


    Results of Colony PCR

    Investigator: Hanna

    Comment: In order to find out, whether the VP2/3 or the VP2/3_Capins fragment was successfully cloned into pCerulean_Zegfr:1907_"Linker" (--> Linker library), pCerulean_6xHis_MiddleLinker and pCerulean_CFP_MiddleLinker, a colony PCR was performed yesterday. From each plate 2-4 clones were picked, so that we got 41 samples (including one positive control = pAAV_RC). With VP_3500_for and VP_4200_rev primer a 880 fragment should be amplified of each colony, if cloning functioned.

    Samples were loaded onto a 1% agarose gel:

    • 1. pCerulean_Zegfr:1907_ShortLinker_VP2/3
    • 2. pCerulean_Zegfr:1907_MiddleLinker_VP2/3_Capins
    • 3. pCerulean_Zegfr:1907_LongLinker_VP2/3
    • 4. pCerulean_6xHis_MiddleLinker_VP2/3
    • 5. pCerulean_Zegfr:1907_SEG_VP2/3
    • 6. pCerulean_6xHis_MiddleLinker_VP2/3_Capins
    • 7. pCerulean_CFP_MiddleLinker_VP2/3_Capins
    • 8. pCerulean_Zegfr:1907_ShortLinker_VP2/3_Capins
    • 9. pCerulean_ Zegfr:1907_SEG_VP2/3_Capins
    • 10. pCerulean_Zegfr:1907_LongLinker_VP2/3_Capins
    • 11. pCerulean_Zegfr:1907_MiddleLinker_VP2/3


    Freiburg10 ColonyPCR15 09 10.png

    As can be seen in the gel picture, it seemed that just clone 9.3, 10.2, 11.1 and 11.2 were VP2/3 positive. Curiously the positive control (K) was negative.

    These 4 clones were mini-preped by Achim and sent for sequencing to GATC: primer = GATC_std_CMV-F.

    Because I found out that some things went wrong during VP cloning due to false geneious annotations and theoretical cloning approaches, all pAAV_RC constructs (with/without Cap or Rep-insert, 4-fold mutated, PvuII insertion, KpnI-backmutation,...) were theoretically prepared again (see geneious folder: "pAAV_RC constructs (DO NOT CHANGE!!!)").
    In addition to that the theoretical cloning of the final N-terminal fusion approaches to VP2/3 were performed once again with these correct sequences. With the help of Sven cloning of the not yet positive approaches were repeated (The ones which were negative in colony PCR).

    To do (tomorrow): Pciking clones, analyze sequencing. Check and plan HSPG-KO approaches and Zegfr:1907

    Practical Cloning:


      • Vector: pCerulean_CFP_MiddleLinker (P407), pCerulean_Zegfr:1907_MiddleLinker (P408), pCerulean_Zegfr:1907_SEG (P409), pCerulean_Zegfr:1907_ShortLinker (P410), pCerulean_Zegfr:1907_LongLinker (P412), pCerulean_6xHis_MiddleLinker (P374)
      • Insert: pSB1C3_VP2/3 (P440), pSB1C3_VP2/3_ins_cap (P444)
      • new vector name: pCerulean_CFP_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_SEG_VP2/3, pCerulean_Zegfr:1907_ShortLinker_VP2/3, pCerulean_Zegfr:1907_LongLinker_VP2/3, pCerulean_6xHis_MiddleLinker_VP2/3 AND pCerulean_CFP_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_SEG_VP2/3-insCap, pCerulean_Zegfr:1907_ShortLinker_VP2/3-insCap, pCerulean_Zegfr:1907_LongLinker_VP2/3-insCap, pCerulean_6xHis_MiddleLinker_VP2/3-insCap
    • buffer used:4

    Comment Because the VP2/3 frament has approximately the same size as the backbone pSB1C3 it may be difficult to separate them on the agarose gel. Therefore a single cutter enzyme (MscI) was chosen to digests the backbone. This enzyme also works in buffer 4 + BSA and needs to be incubated at 37°C.

      • Vector: AgeI, SpeI
      • Insert: NgoMIV, SpeI, SacI



    Digestion


    Freiburg10 Digestion16 09003.jpg

    • Incubation: 2 h


    Agarose gel


    Green boxes were cut out.


    Gelex, ligation and trafo were performed according to standard protocol.
    Next step: Colony PCR of each ligation approach.

    122. labday 17.09.2010

    Colony PCR of N-terminal fusion approach

    Investigator: Achim

    Comment:

    Freiburg10 17 9 colonyPCR.png

    Sequencing analysis of pSB1C3_VP123

    Investigator: Bea

    Comment: The constrcuct was created by performing a PCR with the pAAV_RC_inscap construct. The sequence analysis reveals that the suffix looks well. Additionally the mutation from TAA to TGA can be seen.

    Freiburg10 Seqanalysis pSB1C3 VP123 inscap.jpg

    ViralBricks: Picking clones from T4 ligation

    Investigator: Achim


    Sequencing results N-terminal fusion - part 1

    Investigator: Hanna

    Comment: Colony PCR delivered that 4 clones contained the VP2/3 or VP2/3_Capins sequence. These clones were sent for sequencing:

    1. pCerulean_Zegfr:1907_LongLinker__VP2/3_Capins:

    Freiburg10 pCer LL Aff.png


    Sequencing looked well: VP2/3_Capins was successfully fused to the C-terminus of the Long-Linker. The additional basepair at the beginning of the Affibody was misinterpreted (as one can see in the chromatogram) by Geneious.

    2. pCerulean_Zegfr:1907_MiddleLinker__VP2/3 clone 1:

    Freiburg10 pCer ML Aff.png


    Sequencing looked well: VP2/3 was successfully fused to the C-terminus of the Middle-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious.

    3. pCerulean_Zegfr:1907_MiddleLinker__VP2/3 clone 2:

    Freiburg10 pCer ML Aff 2.png


    Sequencing looked well: VP2/3 was successfully fused to the C-terminus of the Middle-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious.

    4. pCerulean_Zegfr:1907_SEG__VP2/3_Capins:

    Freiburg10 pCer SEG Aff.png


    Sequencing looked well: VP2/3_Capins was successfully fused to the C-terminus of the SEG-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious.


    Mini-Prep and test digestion of several constructs

    Investigator: Stefan

    Glycerol stocks were prepared:

    • B430 = pSB1C3_001_RC_4fold mutated clone 1
    • B431 = pSB1C3_001_RC_4fold mutated clone 2
    • B432 = psB1C3_001_RC_InsRepCap clone 1
    • B433 = psB1C3_001_RC_InsRepCap clone 2
    • B434 = pSB1C3_001_RC_InsCap clone 1
    • B435 = pSB1C3_001_RC_InsCap clone 2
    • B436 = pSB1C3_001_RC_InsRepCap_KpnIback clone 1
    • B437 = pSB1C3_001_RC_InsRepCap_KpnIback clone 2
    • B438 = pSB1C3_001_VP2/3insCap clone 1
    • B439 = pSB1C3_001_VP2/3insCap clone 2
    • B440 = pSB1C3_ZEGFR:1907_VP2/3 clone 1
    • B441 = pSB1C3_ZEGFR:1907_VP2/3 clone 2
    • B442 = pSB1C3_GFP_RFC25_upper band clone 1
    • B443 = pSB1C3_GFP_RFC25_upper band clone 2
    • B444 = pSB1C3_GFP_RFC25_lower band clone 1
    • B445 = pSB1C3_GFP_RFC25_lower band clone 2
    • B446 = pSB1C3_GFP_RFC25 clone 1
    • B447 = pSB1C3_GFP_RFC25 clone 2



    Mini-Prep was performed according to the standard protocol

    • P506 pSB1C3_001_RC_4fold mutated clone 1
    • P507 pSB1C3_001_RC_4fold mutated clone 2
    • P508 psB1C3_001_RC_InsRepCap clone 1
    • P509 psB1C3_001_RC_InsRepCap clone 2
    • P510 pSB1C3_001_RC_InsCap clone 1
    • P511 pSB1C3_001_RC_InsCap clone 2
    • P512 pSB1C3_001_RC_InsRepCap_KpnIback clone 1
    • P513 pSB1C3_001_RC_InsRepCap_KpnIback clone 2
    • P514 pSB1C3_001_VP2/3insCap clone 1
    • P515 pSB1C3_001_VP2/3insCap clone 2
    • P516 pSB1C3_ZEGFR:1907_VP2/3 clone 1
    • P517 pSB1C3_ZEGFR:1907_VP2/3 clone 2
    • P518 pSB1C3_GFP_RFC25_upper band clone 1
    • P519 pSB1C3_GFP_RFC25_upper band clone 2
    • P520 pSB1C3_GFP_RFC25_lower band clone 1
    • P521 pSB1C3_GFP_RFC25_lower band clone 2
    • P522 pSB1C3_GFP_RFC25 clone 1
    • P523 pSB1C3_GFP_RFC25 clone 2
    • P524 pSB1C3_VP2/3
    • P525 pSB1C3_VP2/3_ins_cap



    Test digestion:
    Test digestion was performed for plasmids P506, P507, P510 to P513 and for P522 and P523. Test digestion of P514 to P521 will be performed tomorrow. Preps for P524 and P525 were inocculated from glycerol stock and therefor no digestion will be performed. The constructs were digested using XbaI and AgeI for 40 minutes at 37°C.


    components P470-P481P470-P481
    DNA 2 2
    Buffer 4 (10x)1 1
    XbaI 0,4 0,4
    AgeI 0,40,4
    H2O7,26,2
    Total volume 10 10


    Preparation of gel:

    • 0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time: 40 minutes


    Freiburg10 test digestion pSB1C3 001 RepCaps pSB1C3 GFP RFC25.jpg

    Comment:

    Sent for sequencing:
    Primer used:
    VR2
    plasmid no:
    P522
    tube labeled: SB_1

    Sent for sequencing

    Investigator: Stefan
    pSB1C3_SV40 (P493) was sent for sequencing using VR2 primer. Results have to be checked tomorrow.


    Sequencing analysis of VP2/3 insertion into pCerulean_VP1up_NLS_targeting motif

    Investigator: Stefan
    Comment: Sequences can be found in GATC Watchbox (date 15.10.10) labeled SB_1 (VP2/3 insertion) and SB_2 (VP2/3_insCap insertion).

    Freiburg10 Sequencing VP2 3 insertion.JPG


    Freiburg10 Sequencing VP2 3 insCap insertion.JPG


    Biobrick production of VP2/3- and VP2/3 cap-Gsat_linker

    Motivation: Production of fusion proteins

    Investigator: Kira

    Digestions of backbones and inserts:


    Components VP2/3 VP2/3 cap Gsat linker
    DNA 7 7 7
    BSA (100x) 0 0 0
    Buffer no. 4 (10x) 2,5 2,5 2,5
    EcoRI 0,5 0,5 0,5
    NgoMIV 0,5 0,5 0
    XbaI 0 0 1,25
    H2O 14,5 14,5 13,75
    Total volume 25 25 25


    incubation @ 37 C for approx. 3 h

    1% agarose gel

    Freiburg10 2010 09 18.jpg


    Ligation:

    T4 ligase was used. ligation over night @ 18 C

    VP2/3-mix 8 ul (3,7 ul vector + 4,3 ul insert)
    VP2/3 cap-mix 8 ul (3,7 ul vector + 4,3 ul insert)

    123. labday 18.09.2010

    Continuation of preparation of competent E.coli

    Investigator: Jessica
    preparation of competent XL1blue was finished according to the standard protocol

    • aliquots of 60µl (to use for 1 trafo) are stored in -80°C freezer

    don't use before results of testtrafo is writen down

    Test digestion of pSB1C3_ZEGFR:1907_VP2/3

    Investigator: Jessica


    Samples Plasmid Clone Concentration [ng/µl]
    P514 pSB1C3_001_VP2/3insCap 1 318.9
    P515 pSB1C3_001_VP2/3insCap 2 321.8
    P516 pSB1C3_ZEGFR:1907_VP2/3 1 191,6
    P517  pSB1C3_ZEGFR:1907_VP2/3 2210,8
    P518  pSB1C3_GFP_RFC25_upper band 1138.3
    P519 pSB1C3_GFP_RFC25_upper band 272.3
    P520 pSB1C3_GFP_RFC25_lower band 1141.6
    P521  pSB1C3_GFP_RFC25_lower band 2127.5


    Test digestion:

    components Volume for each sample P516/517 /µl Volume for each sample P514/515 /µlVolume for each sample P518-521 /µl
    DNA 2,5 3,5 1,5
    BSA (10x) 1 1 1
    Buffer 1/4/4 (10x) 1 1 1
    Enzyme NgoMIV 0,5 - -
    Enzyme DraIII 0,5 - -
    Enzyme XbaI - - 0,4
    Enzyme AgeI - - 0,4
    Enzyme MscI - 0,4 -
    Enzyme SpeI - 0,4 -
    H2O 4,5 3,7 5,7
    Total volume /µl 10 10 10


    Preparation of gel:

    • Incubation time: 1,5 h, Incubation temperature: 37°
    • 0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt



    Freiburg10 Test digestion P514-521.jpg

    • Expected fragment sizes:

    P516/517: 3540bp / 651bp
    P514/515: 1300bp / 2700bp
    P518-521: 2000bp / 700bp


    Comment: All samples look OK! Samples will be sent for sequencing on monday.

    Mini-Prep of pSB1C3_SV40 clone 1 (B420)

    Investigator: Jessica
    was done because of the low concentration of the plasmid P493

    • P526: pSB1C3_SV40 clone 1 (=B420) c= 176,0 ng/µl

    New aliquots (60µl) of BL21 can be used ! (Jessica)



    New Colony PCR of N-terminal fusion approach

    Investigator: Hanna


    Comment: Because just 3 clones of the colony PCR (yesterday) were VP2/3 positive, new colonies were picked for a colony PCR. We assumed that we can find some positive clones, because the 3 successful ligations contained VP2/3 AND VP2/3_Capins, so that we concluded, that digestion worked and that we just have to search for positive clones on the plates.

    A mastermix for 28 samples was prepared:

    • 10x Taq Buffer: 28 µL
    • MgCl2 (50 mM): 2.8 µL
    • dNTPs: 5.6 µL
    • Primer 1 (4200 rev = O42): 5.6 µL
    • Primer 2 (3500 for = O39): 5.6 µL
    • Taq polymerase: 1.4 µL
    • H2O: 231 µL

    --> Volume for each reaction: 10 µL

    4 colonies were picked with a tooth-pick from each plate (6), inoculated into the 10 µL mastermix and then put into 5 µL DYT containing Kanamycin.
    As positive control 1 ng of P158 = pAAV_RC_SDM_SalI and as negative control 1 ng of P42 = pAAV_RFC25 were used.

    PCR program:

    • 95°C, 6'

    30x

    • 95°C, 25
    • 50.5°C, 30
    • 68°C, 54

    1x

    • 68°C, 5'

    Hold 4°C

    Gel picture:

    Freiburg1 CPCR allpos002.jpg


    All PCR reactions delivered positive VP2/3 results (expected size: 880 bp). Unfortunately also the negative control was positive. We decided that 2 clones of each plate will be mini-preped. The remaining 2 clones will be pelletized and stored at -20°C. On monday several test digestions will be performed (perhaps 1 of each plate).

    Trafo of pSB1C3_GSAT_VP2/3 and pSB1C3_GSAT_VP2/3_Capins

    Investigator: Hanna


    Comment: Because we just used the Long-, Middle-, Short- and SEG-Linker for the small linker library, we decided also to perform the N-terminal fusion to VP2/3 with the GSAT linker.
    Yesterday, VP2/3 and VP2/3_Capins were cloned to the C-terminus of the GSAT linker. Ligation was performed over night. New plasmids: pSB1C3_GSAT_VP2/3 and pSB1C3_GSAT_VP2/3_Capins
    Trafo was performed according to standard protocol: XL1b cells, 2 µL DNA. Plates will be stored over night @ 37°C room.
    To do tomorrow: Picking clones.


    Colony-PCR of pSB1C3_CD

    Investigator: Bea

    Comment: Since the colony PCR of the last try did not work exactly the way we expected it, another colony PCR approach with new primers was conducted.


    The protocol:

    • Forward primer used: iGEM RFC25
    • Reverse primer used: iGEM RFC25
    • Cells: XL1-Blue


    A mastermix for 37 samples was prepared for using it in this approach and the colony PCR for the loop insertion motif Z34C.

    • 10x Taq Buffer: 1.0 µL
    • MgCl2 (50 mM): 0.1 µL
    • dNTPs: 0.2 µL
    • Primer iGEM RFC25 forward: 0.2 µL
    • Primer iGEM RFC25 reverse: 0.2 µL
    • Taq polymerase: 1.4 µL
    • H2O: 231 µL
    • Total volume for each reaction: 10 µL


    PCR programm used:



    Corresponding inoculated cells put in the 37°C room over night at 21:00.

    BioBrick Assembly of ITR_phTERT_betaglobin_mVenus to hGH_rightITR

    Investigator: Bea

    Comment: For assembling the last BioBricks together in order to obtain the fully assembled vectorplasmid which contains the tumorspecific promoter phTERT and the GOI mVenus.


    Digestion of the constructs:

    • P485 = pSB1C3_leftITR_phTERT_betaglobin_mVenus c=257 ng/µL
    • P440 = pSB1C3_hGH_rightITR c=105 ng/µL


    Components vP485 /µL vP186/µL
    DNA 4 14
    BSA (10x) 2 2.5
    Buffer no. 4 (10x) 2 2.5
    EcoRI 1 1
    AgeI 1 1
    H2O 10 4
    Total volume 20 25


    The expected fragments of the digested constructs were: The underlined fragments represent the fragments which correspond to the desired construct which can be used for ligation.

    • P485: 3911bp
    • P186: 657, 2072bp


    Loading plan:

    M    P485    P186
    


    Results:

    Freiburg10 pSB1C3 phTERT mVenus 18 09 2010.jpg


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

    Ligation:

    • v=P485 =4,96µL
    • v=P309 =3,04µL

    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.

    Colony-PCR of ViralBricks

    Investigator: Achim

    Comment: We decided to screen the new ViralBrick clones for the right insert via colony-pcr. This way we avoid unnecessary minipreps of clones containing religated vector or BLA.


    The protocol:

    • Forward primer used: iGEM RFC25
    • Reverse primer used: iGEM RFC25
    • Cells: XL1-Blue


    A mastermix for 37 samples was prepared for using it in this approach and the colony PCR for the loop insertion motif Z34C.

    • 10x Taq Buffer: 1.0 µL
    • MgCl2 (50 mM): 0.1 µL
    • dNTPs: 0.2 µL
    • Primer iGEM RFC25 forward: 0.2 µL
    • Primer iGEM RFC25 reverse: 0.2 µL
    • Taq polymerase: 1.4 µL
    • H2O: 231 µL
    • Total volume for each reaction: 10 µL


    PCR programm used:

    Corresponding inoculated cells put in the 37°C room over night at 21:00.

    => Go to Labjournal September part 2 (labday 124 - 135)