Team:Freiburg Bioware/NoteBook/Labjournal/September

• 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

2. pCerulean_6xHis_MiddleLinker

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:

• Incubation: 55 minutes

Agarose-Gel:

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

• Marker: GeneRuler ladder mix

Comments: Test digestion looked well:


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:

Mini-Prep and test digestion of the CD biobricks

Investigator: Kira

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??

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.

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.

2. pCerulean_6xHis_MiddleLinker: Sequencing looked also well.

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

• Incubation: 1.5 h

Agarose-Gel:

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

• Marker: GeneRuler ladder mix

Gel extraction

Gel measurement:

Ligation

1. PCerulean_Zegfr:1907_LongLinker

2. PCerulean_Zegfr:1907_MiddleLinker

3. PCerulean_Zegfr:1907_SEG

4. PCerulean_Zegfr:1907_ShortLinker

5. PCerulean_CFP_MiddleLinker

Trafo

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

Harvest viral particles, Seeding HT1080 cells

Investigator: Adrian, Kerstin

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

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

Investigator: Patrick

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

• P381: pCerulean_middlelinker clone 1 upper band : 526,0 ng/µl
• P382: pCerulean_middlelinker clone 2 upper band : 522,0 ng/µl
• P383: pCerulean_middlelinker clone 3 upper band : 463,0 ng/µl
• P384: pCerulean_middlelinker clone 1 lower band : 465,0 ng/µl
• P385: pCerulean_middlelinker clone 2 lower band : 500,0 ng/µl
• P386: pCerulean_middlelinker clone 3 lower band : 524,0 ng/µl

Öhm Patrick: Hier ist der Grund, warum das Experiment "wiederholt" wurde: Falsche Beschrieftung hier, als auch in der Excel Tabelle ;) "Finde den Fehler!" :P

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

;-) bitte ausfüllen

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

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

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

Results:

• 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

• 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 Z_EGF-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:

• 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

• 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 Z_EGF-R:1907:

v_{Cerulean_VP1up_NLS} = 5,82µL
v_ZEGF-R:1907 = 2,18µL



mVenus Z:

v_{Cerulean_VP1up_NLS} = 4,04µL
v_mVenus = 3,96 µL



6xHisTag:

v_{Cerulean_VP1up_NLS} = 0,2µL
v_6xHisTag = 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:

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

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:

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

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:

° 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

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:

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:

PCR program:

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).

SL: Shortlinker ML: Middlelinker LL: Longlinker

Test digestion:

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

• 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

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

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

Sent for sequencing

Investigator: Stefan

• Tube name: SB2

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

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

113. labday 07.09.2010

Sequencing results of new loop insertion ligations

Investigator: Achim

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

Repetition of CD biobrick production

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

Investigator: Kira
DNA sample was diluted 1:100

PCR program:

Digestion of plasmid backbone:

c (pSB1C3) = 151, 1 ng/ µl

incubation @ 37 C for approx. 2 h

1% agarose gel

Digestion of PCR product:

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

Test digestion:

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

• 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.

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:

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

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:

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

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

Preparation for Midi-Preps of pAAV_mVenus, pHelper and pAAV_RC

Investigator: Kerstin

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

Sequencing

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

114. labday 08.09.2010

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

Investigator: Hanna

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

Sequencing results:
1. pCerulean_CFP_MiddleLinker:

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

2. pCerulean_Zegfr:1907_ShortLinker:

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:

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:

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:

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)

PCR program:

Digestion of plasmid backbone:

c (pSB1C3_CFP) = 151, 1 ng/ µl

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

Digestion of PCR product:

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:

PCR program:

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

incubation @ 37 C for approx. 2 h

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

Midi-Prep

Investigator: Anissa, Kerstin

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

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

Seeding HT1080, Passaging AAV293 and Transfection

Investigator: Kerstin, Patrick

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

Checking fractions of Äkta run

Investigator: Stefan

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

PCR progam: primers used:

PCR program:

Gel run will have to be performed tomorrow.

115. labday 09.09.2010

Picking clones of leftITR_hTERT_beta-globin and leftITR_CMV_beta-globin biobricks

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

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

new ligation try and transformation of CD-biobrick

Kira

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

Transformation was performed according to the standard protocol

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

Investigator: Achim

• Nomenclature:

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

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

Two new reaction setups for the remaining four ViralBricks:

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

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

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

in a 20 µl reaction.

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

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:

PCR program:

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

incubation @ 37 C for approx. 2h

1% agarose gel

Digestion of PCR product:

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.


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.


pCerulean_VP1up_NLS_Histag: Insert was succesfully cloned into p_Cerulean.

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

incubation @ 37 C for approx. h

1% agarose gel

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

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 T_M 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 T_M= 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:

PCR program:

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

Test digestion:

• 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
  

• 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.

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.

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:

1% agarose gel

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)

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:

Klenow Buffer:

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

Investigator: Anna

• P357 = pAAV_RC
• P396 = pAAV_RC_CapIns_prepSDM clone 3

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:

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

   

   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:

Preparation of gel:

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

• 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

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:

Ingredients:

PCR program:

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.

Gel:
0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, at 90 Volt, running time: ~90 minutes


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:

P40:

PCR program:

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

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:

SV40 PCR program:

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

• 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

no DNA on the gel...

for Cap

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

PCR program:

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

incubation @ 37 C for approx. 2h

1% agarose gel

Digestion of PCR product:

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

incubation @ 37 C for approx. 2h

1% agarose gel

BioBrick ^TM assembly of pSB1C3_leftITR_promoter_betaglobin to several GOIs

Investigator: Bea

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

Digestion of the constructs:

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

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

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

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

• 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.

• Marker: GeneRuler ladder mix

Gel extraction

Gel measurement:

Ligation

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.

3^rdrepetition 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:

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.

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

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:

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

Results:

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:

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

• 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

• Marker: GeneRuler ladder mix

Gel extraction

Gel measurement:

Ligation

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.

Preparation of gel:

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

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:

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