Team:Freiburg Bioware/NoteBook/Labjournal/September2

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September

124. labday 19.09.2010

Continuation with Colony-PCR of pSB1C3_CD and ViralBrick motif Z34C

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.

Loading plan:
upper lanes

M   CD1  CD2  CD3  CD4  CD5  CD6  CD7  CD8  CD9  CD10   +ctrl(CFP) +ctrl(BAP) -ctrl(pAAV_MCS)   11Q1  12Q1  13Q1 13Q2  M

lower lanes

M   11T41  11T42  11T43  11T44    12T41  12T42  12T43  12T44    13T41  13T42  13T43  13T44    14T41  14T42 14T43  14T44  M

Expected sizes of the PCR products are:

• Cytosine deaminase (CD) = 1300bp
• Z34C loops insertion motif = 220bp
• Positive control 1 = CFP = 750bp
• Positive control 2 = BAP_587 = 170bp

Results:
The Colony PCR of the Cytosine Deaminase (CD) did not work out. The detectable bands at around 700 bp correspond to CFP. Therefore, BioBrick production of the CD must be repeated. The following three bands represent the controls. Three different controls were used. The first corresponds to pSB1C3_CFP, the second to BAP_587 and third control is the negative control. Unfortunately now bands can be detected in the postive controls. A faint band can be seen in the negative control which is the only lane in which no band should be detectable.
The following bands after the three controls correspond to the approaches of the Z34C Viralbrick production. Some bands in the lower and the upper lanes show positive results. The corresponding inoculated overnight culture were centrifuged and prepared in order to perform a Mini-Prep tomorrow.

Comment: hmm, seems that the CD-assambly didn't work at all.. Should i try it again or are we gonna skip it for good due to other priorities?(kira)
No, we cannot skip it. Can we change something else?? Any ideas what we can alter in the protocol? Because problem is that we are still waiting for the tk... so we should aswell focus on the other prodrug activating enzmye!! (Bea)

Testtransduction of the final modified capsid coding constructs

Investigator: Adrian

Aim of the experiment:
During the project 22 silent nucleotide exchange mutations were introduced into the capsid coding construct to make it compatible with the RFC standards and to have single cutting restriction enzymes flanking the 453 and the 587 loop sequence. Two point mutations had to be dismissed, because either a first test transduction showed that the construct was not working anymore or the insertion of the synthesized gene posed serious problems, because the restriction enzyme did not work.
Finally, we have a construct that is shown to produce infectious particles comparable to the current AAV systems and carries 20 point mutations. Now we can announce that the Adeno-associated Virus is compatible to the RFC standard and the idea to replace the loop sequences via ViralBricks works!

Impression from the moment:

The construct with the insertion of the Rep and the Cap synthesis worked! Reintroduction of the KpnI restriction site recovered the functionality of the viral genome!

Miniprep of serveral constructs

Investigator: Stefan

Glycerol stocks were prepared:

• B430 = pCerulean_ZEGFR:1907_Longlinker_VP2/3
• B431 = pCerulean_CFP_Middlelinker_VP2/3_insCap
• B432 = pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap

Mini-Prep was performed according to the standard protocol

• P527 = pCerulean_ZEGFR:1907_Longlinker_VP2/3 c = 230,61 ng/µl
• P528 = pCerulean_CFP_Middlelinker_VP2/3_insCap c = 290,81 ng/µl
• P529 = pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap c = 269,39 ng/µl

Several other constructs were preped, but need to be confirmed before being added to plasmid/glycerol stock.

125. labday 20.09.2010

Minipreps and Sequencing of ViralBrick clones

Investigator: Achim

The following clones were prepped:

• Quickligation:
  • 11.1, c = 85,41 ng/µl
  • 13.1, c = 96,93 ng/µl
• T4-Ligation:
  • 11.4, c = 76,42 ng/µl
  • 12.1, c = 80,36 ng/µl
  • 12.4, c = 85,43 ng/µl
  • 13.1, c = 90,06 ng/µl
  • 13.2, c = 80,87 ng/µl
  • 13.4, c = 76,62 ng/µl
  • 14.1, c = 76,50 ng/µl
  • 14.2, c = 98,52 ng/µl

Clones 11.4, 12.1, 13.2, 14.2 (T4) were sent for sequencing.

Cloning of pCerulean_Affibody_VP2/3 and pSB1C3_Affibody_middlelinker_GFP_his

Investigator: Jessica

Comment:Affibody_VP2/3 will be cloned in pCerulean and Affibody_middlelinker will be cloned together with GFP_His in pSB1C3

• P407= pCerulean_CFP_middlelinker c=482,76 ng/µl
• P516= pSB1C3_ZEGFR:1907_VP2/3 clone 1 c= 191,6 ng/µl
• P290= pSB1C3_Affibody_Middlelinker clone 1 c= 227,4 ng/µl
• P518= pSB1C3_GFP_RFC25_upper band clone 1 c= 138,3 ng/µl
• P520= pSB1C3_GFP_RFC25_lower band clone 1 c= 141,6 ng/µl

Digestion:

1,0 g Agarose,100 ml TAE (1%), 6 µl GELRED , at 120 Volt

will be repeated tomorrow

Gelextraction:

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

• P407= c= ng/µl
• P516= c= ng/µl
• P290= c= ng/µl
• P518= c= ng/µl
• P520= c= ng/µl

T4 Ligation:

The Ligation was performed as following:

• Vector Volume: µl
• Insert Volume: 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 (XL1blue). The cells were plated on a agar plate with Kana/Cm

Cloning of P5_TATAless downstream of pSB1C3_001_RC_insrepcap_KpnI_back and P5 upstream of pSB1C3_001_RC_insrepcap_KpnI_back

Investigator: Anissa
Digestion of the constructs:

• P320 = pSB1C3_001_RC_insrepcap_KpnI_back c=408 ng/µL
• P320 = pSB1C3_001_RC_insrepcap_KpnI_back c=408 ng/µL
• P180 = pSB1C3_pTAV2 (P5)clone 1 c=108,8 ng/µL
• P184 = pSB1C3_pAAV_RC (P5TATAless)clone 3 c=176,9 ng/µL

Loading plan:

M    P320(upstream)    P320(downstream)    P180    P184

Results:

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

Ligation:

• v_{=P320_up} =7,37µL
• v_{=P180_up} =0,63µL
• v_{=P320_down} =7µL
• v_{=P184_down} =1µL

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

Next steps:
Picking clones and perform Mini-Prep.

Test digestion of VP2-N-terminal fusion constructs

Investigator: Hanna

Comment: On Satureday a colony PCR was performed of:
1. pCerulean_Zegfr:1907_ShortLinker_VP2/3 (P530)
2. pCerulean_Zegfr:1907_SEG_VP2/3 (P531)
3. pCerulean_CFP_MiddleLinker_VP2/3 (P532)
4. pCerulean_6xHis_MiddleLinker_VP2/3 (P533)
5. pCerulean_6xHis_MiddleLinker_VP2/3_insCap (P534)
6. pCerulean_Zegfr:1907_MiddleLinker_VP2/3_insCap (P535)

Because all constructs (4 clones of each approach), including the negative control (pAAV_RFC25), showed positive results, one test digestion of each construct was performed.

Digestion

--> 2 µL DNA + 8 µL Master Mix

• Incubation: 1.5 h

Agarose-Gel:

0.4 g Agarose, 50 mL TAE (0.8 %), 3 µL GELRED, at 100 Volt, running time: 50 minutes

• Marker: GeneRuler ladder mix

Test Digestion N-terminal VP2 Fusion

Comment: Test digestion delivered positive results: Expected fragment size after VP2/3 fusion to Zegfr:1907_Linker: 2180 bp - compared to failed VP2/3 insertion: ~ 220 bp.
Expected fragment size after VP2/3 fusion to CFP_MiddleLinker: 2710 bp - compared to failed VP2/3 insertion: ~ 760 bp.
pCerulean_ZEGFR:1907_Longlinker_VP2/3
pCerulean_CFP_Middlelinker_VP2/3_insCap
pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap
pCerulean_ZEGFR:1907_Shortlinker_VP2/3
pCerulean_ZEGFR:1907_SEG_VP2/3
pCerulean_CFP_MiddleLinker_VP2/3
pCerulean_6xHis_MiddleLinker_VP2/3
pCerulean_6xHis_MiddleLinker_VP2/3_insCap
pCerulean_ZEGFR:1907_MiddleLinker_VP2/3_insCap
will be sent for sequencing to GATC.