Team:Freiburg Bioware/NoteBook/Labjournal/September2

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<b>Comment:</b> Sequences looked well. The 6 samples with VP2/3_insCap will be tested today in cell culture.
<b>Comment:</b> Sequences looked well. The 6 samples with VP2/3_insCap will be tested today in cell culture.
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Revision as of 13:35, 22 September 2010

Contents

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.

Freiburg10 ColonyPCR Results 19.09.2010.jpg



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

Picking the clones and preparing the over-night culture

Investigator: Kira

VP2/3_Gsat-linker as well as VP2/3_cap_Gsat-linker plates contain colonies, thus 3 clones from each plate were inoculated into 5 ml DYT+ 5ul Chloramphenicol and incubated @ 37 C

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:

components P407 P516 P290 P518 P520
DNA 3,5 10 7 10 10
BSA (10x) 2 2 2 2 2
Buffer 4 (10x)2 2 2 2 2
EcoRI 0,8 0,8---
AgeI 0,80,80,8--
NgoMIV ---0,80,8
SpeI --0,80,80,8
H2O10,9 4,4 7,4 4,4 4,4
Total volume 20 20 20 20 20


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

P516, P518, P520 will be repeated tomorrow (no DNA after gel-ex)

Freiburg10 pCerulean affi vp23.jpg




Gelextraction:

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

  • P407 c= 6,5 ng/µl
  • P516 c= - ng/µl
  • P290 c= 7,3 ng/µl
  • P518 c= - ng/µl
  • P520 c= - ng/µl


stands in 4°C room for comtinuation tomorrow

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


Components vP320 upstream /µL vP180 upstream/µL vP320 downsteam /µL vP184 downsteam/µL
DNA 2,513,8 2,5 8,5
BSA (10x) 1,52 1,5 1,5
Buffer no. 4 (10x) 1,52 1,5 1,5
Enzyme 1 XbaI 1SpeI 1 SpeI 1XbaI 1
Enzyme 2 EcoRI 1EcoRI 1 PstI 1PstI 1
H2O 7,50,27,51,5
Total volume 15 20 15 15


Loading plan: 

M    P320(upstream)    P320(downstream)    P180    P184


Results:

File:.jpg
500px


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

components Components Master Mix /µl
BSA (10x) 7
Buffer 4 (10x) 7
EcoRI 3.5
SpeI 3.5
H2O 35

--> 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

Sample Sample/µl] Loading dye (6x)/µl Expected size 1 Expected size 2
P 530 10 µl 2 µl 2710 bp 3937 bp
P 531 10 µl 2 µl 2710 bp 3937 bp
P 532 10 µl 2 µl 2710 bp 3937 bp
P 533 10 µl 2 µl 2710 bp 3937 bp
P 534 10 µl 2 µl 2710 bp 3937 bp
P 535 10 µl 2 µl 2710 bp 3937 bp


  • Marker: GeneRuler ladder mix
Marker /µL Sample P530 /µl Sample P531 /µl Sample P532 /µl Sample P533 /µl Sample P534 /µl Sample P535 /µl
Lane 4 12 12 12 12 12 12



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.

BioBrick production: PCR of pAAV_RC_InsRepCap_KpnIback_VP1-ko and VP2-ko

Investigator: Stefan

DNA samples were diluted 1:1000

  • P455 = pAAV_RC_1.2 SDM SalI
  • P463 = pAAV_RC_CapIns_prepSDM clone 3

PCR progam:

Ingredients Volume P455 / µl Volume P463 / µl
5X Phusion HF buffer 10 10
10 mM dNTP mix1 1
forward primer: O93 2,5 2,5
reverse primer: O115 2,5 2,5
DNA Template33
DMSO 2 2
Phusion Polymerase0,5 0,5
H2O28,5 28,5
Total volume50 50



PCR program:

CyclesTemperature / °CTime / s
19860
2 ( step 2-4: 8x)9815
35925
47275
5 (step 5-6: 17x)9815
67285
7 72300
Hold 4 4


Gel:
0,5 g agarose (1%), 50 ml TAE, 3µl GELRED running time: 45 minutes at 110V

Freiburg10 RepCap VP1 2 ko PCR.jpg

Gel-Extraction:
Gel-extraction was performed according to standard protocol. The complete amount eluted was used for PCR digestion.


Digestion of PCR product:

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


Digestion was performed at 37 °C for 2 hours.

PCR-Purification:
Gel-extraction was performed according to standard protocol.

  • c (P455) = 13,5 ng/µl
  • c (P463) = ng/µl

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

Components vector Volume/µL
DNA 3
BSA (10x) 2
Buffer no. 4 (10x) 2
EcoRI 1
SpeI 1
H2O 11
Total volume 20


Digestion was performed at 37 °C for 2 hours.


Gel:
0,5 g agarose (1%), 50 ml TAE, 3µl GELRED running time: 50 minutes at 120V Freiburg10 digestion pSB1C3 001.jpg

Gel-Extraction:
Gel-extraction was performed according to standard protocol.
c(P320) = 0,8 ng/µl

Ligation:

  • 1 µl T4 DNA ligase
  • 1 µl 10x Buffer
  • 8 µl DNA mix (vector + insert)

Amounts of DNA used:

  • c(P320) = 7,73 µl
  • c(P455) = 0,27 µl
  • c(P320) = 7,19 µl
  • c(P463) = 0,81 µl

Incubation time: 45 minutes at room temperature.

Transformation:
Transformation was performed according to standard protocol using XL1b cells and Chloramphenicol as antibiotic.

2x repetition of CD biobrick

Investigator: Kira

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 Template (1:100 dil)0,5 µl
DMSO 0 µl
Phusion Polymerase0,5 µl
H2O33µl
Total volume50 µl


PCR program:

CyclesTemperatureTime
98°C1
10x98°C15"
58°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. 6 h

1% agarose gel

Freiburg10 CD 2010 09 21.jpg

According to the gel results, PCR was repeated twice.. But both tries revealed the same unexpected results. Insert from the last cloning procedure was found and used for the ligation and further transformation.


Ligation
T4 ligase was used
1 ul T4 Buffer
1 ul T4 Ligase
8 ul (0,9 ul vector+ 7,1 ul insert) DNA-mix

incubation @ RT for 45 min

Transformation was performed according to the standard protocol and the cells were spread on the agar plate containing Chloramphenicol.

126. labday 21.09.2010

Trafo evalutation of CD biobrick plate

Investigator: Kira

the agar plate was checked at approx. 4 pm and there were no colonies visible. Taking in account that the plate was incubated from 10.30 pm till 8 am @ 37C due to the electricity failure, the plate was put back @ 37C at 4.30 pm

Comment: the plate was picked this morning by a lab member and put in the coldroom

Continuation of 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:

components P516 P518 P520
DNA 10 10 10
BSA (10x) 2 2 2
Buffer 4 (10x) 2 2 2
EcoRI 0,8--
AgeI 0,8--
MscI 0,8--
NgoMIV -0,80,8
SpeI -0,80,8
H2O 3,6 4,4 4,4
Total volume 20 20 20


0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 120 Volt



Freiburg10 pCerulean affi vp23.2.jpg




Gelextraction:

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

  • P516= 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

Sequenzing result of pSB1C3_EGFP_His upper and (!) lower band

Investigator: Jessica
1. pSB1C3_EGFP_His suffix:

Freiburg10 GFPHis.jpg

2. pSB1C3_EGFP_His prefix:

Freiburg10 GFPHis2.jpg

results look in both plasmids and are used for cloning of pSB1C3_Affibody_middlelinker_GFP_His

Midi-Prep of pHelper & pSB1C3_leftITR_CMV_beta-globin_mVenus_hGH_rightITR

Investigators: Chris W.

Midi-Preps of pHelper=P540 and pSB1C3_leftITR_CMV_beta-globin_mVenus_hGH_rightITR=P541


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

plasmid-no. P540P541
concentration (ng/µl)480,45 1264,20


New aliquots (60µl) of XL1blue can be used ! (Jessica)






127. labday 22.09.2010

PCR of P40

Investigator: Jessica
DNA sample P432 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: O188 2,5
DNA Template3,0
DMSO -
Phusion Polymerase0,5
H2O31
Total volume50,5


PCR program:

CyclesTemperature / °CTime / s
9860
8x9815
6125
728
17x9815
6925
728
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

Freiburg10 pSb1C3 P40.jpg

Comment:

Mini-Prep and test digestion of pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko

Investigator: Stefan

Comment:


Glycerol stocks were prepared:

  • B463 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 1
  • B464 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 2


Mini-Prep was performed according to standard protocol:

  • P548 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 1 c = 435,82 ng/µl
  • P549 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 2 c = 409,21 ng/µl

Test digestion:

Results:
500px

Cloning of CMV promotor into pSB1C3_VP123_inscap

Investigator: Anna

  • Vector: name: pSB1C3_VP123_inscap P489
  • Insert: name: pSB1C3_CMV P193
  • new vector name: pSB1C3_CMV_VP123_inscap P
  • buffer used: 4
  • DNA concentration (vector): 350,0 ng/µl ; DNA concentration (insert): 333,5 µg/µl


components volume of pSB1C3_VP123_inscap /µl volume of pSB1C3_CMV /µl
DNA 3 4,5
BSA (10x) 1,5 1,5
Buffer 4 (10x)1,51,5
Enzyme IEcoI HF 1 EcoI HF 1
Enzyme IIXbaI 1 SpeI 1
H2O7 5,5
Total volume (e.g. 15,20,25,30 µl) 1515


0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time:45


Loading plan for agarose gel:
Loading dye with SDS (6X), 3 µl
Marker used: GeneRuler ladder mix (Fermentas), 4 µl

Marker Sample 489, 18µl Sample 193, 18µl
Lane 1 3 5
Fragment size 4404 bp 681 bp



Freiburg10 Cloning CMV into pSB1C3 VP123 inscap.jpg


Sequencing of Zegfr:1907-Linker Library, His-Tag and imaging appoaches

Investigator: Hanna

Comment: After several problems were managed (cloning difficulties, colony PCR troubles, interchanges of samples,...), sequences of the final constructs needed to be verified in order to test the constructs in cell culture.

Sample gallery

pCerulean_CFP_MiddleLinker_ VP2/3_InsCap (P528) forward

pCerulean_CFP_MiddleLinker_ VP2/3_InsCap (P528) reverse

pCerulean_ZEGFR:1907_ShortLinker_ VP2/3_InsCap (P527) forward

pCerulean_ZEGFR:1907_ShortLinker_ VP2/3_InsCap (P527) reverse

pCerulean_6xHis_MiddleLinker_ VP2/3 (P533) forward

pCerulean_6xHis_MiddleLinker_ VP2/3 (P533) reverse

pCerulean_6xHis_MiddleLinker_ VP2/3_insCap (P534) forward

pCerulean_6xHis_MiddleLinker_ VP2/3_insCap (P534) reverse

pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_insCap (P535) forward

pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_insCap (P535) reverse

pCerulean_ZEGFR:1907_SEG_ VP2/3_insCap (P502) reverse

pCerulean_ZEGFR:1907_LongLinker_ VP2/3_insCap (P503) reverse

pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3 (P504) reverse



Comment: Sequences looked well. The 6 samples with VP2/3_insCap will be tested today in cell culture.

Retrieved from "http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2"