Team:Freiburg Bioware/NoteBook/Labjournal/August2

The cells were transfected with 40µg YFP, 3,3µg Rep/Cap and 3,3µg pHelper.

Comments: We want to find out, if the efficiency of virus production can be influenced by the amount of transfected cells.

Plate 1 YFP: 200.000 cells per well

Plate 2 YFP: 200.000 cells per well

Plate 3 YFP: 200.000 cells per well

Plate 4 YFP: 200.000 cells per well

Plate 5 YFP: 200.000 cells per well

New ladder mix and testing

Investigator: Jessica
new Generuler ladder mix from Fermentas arrived
one tube (containing 100µl solution) was prepared with 100µl loading dye and 400 µl miliporewater

two test-gels were prepared:

• 1% TAE agarose gel with samples of
• 0,5 µl
• 1,0 µl
• 2,0 µl
• 3,0 µl
• 4,0 µl
• 5,0 µl
• 6,0 µl
• 7,0 µl
• 8,0 µl
• 9,0 µl
• 10,0 µl
• sample P250

• 1% TAE agarose gel containing 20% H2O with samples of ( 20% H2O was an idea I have read in a journal but it doesn't seem like a good idea
• 0,5 µl
• 1,0 µl
• 2,0 µl
• 3,0 µl
• 4,0 µl
• 5,0 µl
• 6,0 µl
• 7,0 µl
• 8,0 µl
• 9,0 µl
• 10,0 µl
• sample P251

Comments: an example of these laddervolumes is patches at the bench (right up)

SDM of CD NgoMIV

Investigator:Kira

Comment: Lightning kit was used for quickchange reaction

PCR

sample was digested with 1 ul DpnI for 10 min at 37°C

Transformation was performed according to the standard protocol.

Picking clones of pSB1C3_affibody and pSB1C3_Rep68

Investigator: Anna

°: 2 clones °°: 3 clones each

Clones were inoculated in 10 mL DYT medium containing 10 µL of antibiotics and were put in 37°C room on shaker.

95. labday 20.08.2010

Sequence analysis of sequencing reads prepared yesterday (19.08.2010)

Investigator: Bea

GENERAL COMMENT: pSB1C3 {Rep 52 and Rep 78{} were sent for sequencing BUT they cannot be used for subcloning the ordered rep protein because in the produced BioBricks the restriction enzyme needed for subcloning (SwaI) is NOT present in the BioBricks pSB1C3_Rep52 and rep78.

Comments: Sequence analysis of pAAV_RC containing the subcloned "rep" (ordered)

• Primer used: VP1 primer fro pKex
• Plasmid sequenced: P250
• Sequence sample: CL-9
• Stored in folder:

Next steps: xy

Comments: Sequence analysis of pAAV_RC containing the subcloned "rep" (ordered)

• Primer used: VP1 primer fro pKex
• Plasmid sequenced: P251
• Sequence sample: CL-10
• Stored in folder:

Next steps: xy

Comments: Sequence analysis of pSB1C3_Rep40

• Primer used: VR-2
• Plasmid sequenced: P231
• Sequence sample: CL-2
• Stored in folder: DKFZ Rep40ex

Sequence read is good (prefix is ok) but there was found a mutation in Rep52 protein: G2886A (nucleotides) --> V337I.

Next steps: xy

Comments: Sequence analysis of pSB1C3_AAP

• Primer used: VR-2
• Plasmid sequenced: P239
• Sequence sample: CL-3
• Stored in folder: DKFZ AAP

xy

Next steps: xy

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Continuation: pSB1C3_SDM_SspI_Bla14FM clone 5 SDM (P222)

Investigator: Patrick

The XL1B transformation didnt work or was too much diluted because there were no clones on the plates. The dilution steps of the transformed XL10-Gold cells worked quite well. Now there are 'pickable' clones.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Subcloning cap from pMA_RepCap into pAAV_RC which contains four mutations and the synthesized Rep part

Investigator: Patrick

pAAV_RC_ins-rep clone 1 (P250) and pMA_RepCap Vector_SDM_InsPvuII clone 1 (P211) were digested with PspLI and BspMI according to the standard protocol (Subcloning_cap_into_pAAV_RC).

Expected size of the bands:

• Insert: 920 bp (P211)
• Vector: 6414 bp (P250)

The gelextraction yielded the following DNA concentrations:

• P 211 : 2,45 ng/µl
• P 250 : 11,15 ng/µl

The marked bands were cut out and the DNA was extracted. The ligation was performed accodring to the standard prtocol with a total volume of 10 µl: 2,67 µl vector backbone(P250), 5,33 µl Insert following a transformation according to the standard protocol with XL1B cells.

Mini-Preps of pSB1C3_Rep68_ex and pSB1C3_ZEGFR:1907

Investigator: Chris L.,Stefan

Glycerol stocks were prepared:
• B218 = pSB1C3_Rep68_ex clone1
• B219 = pSB1C3_Rep68_ex clone2
• B220 = pSB1C3_Rep68_ex clone3




• B221 = pSB1C3_ZEGFR:1907 clone 1
• B222 = pSB1C3_ZEGFR:1907 clone 2


Mini-Prep following the standard protocol


• B218 = pSB1C3_Rep68_ex clone1 c=286.72 ng/µl
• B219 = pSB1C3_Rep68_ex clone2 c=277.06 ng/µl
• B220 = pSB1C3_Rep68_ex clone3 c=298.18 ng/µl






• B221 = pSB1C3_ZEGFR:1907 clone 1 c=174.20 ng/µl
• B222 = pSB1C3_ZEGFR:1907 clone 2 c=162.48 ng/µl
  
  

Test digestion

• buffer used: 4 ; Restriction-enzymes used: XbaI ; SpeI ; EcoRI ; PstI




Components	P264 Volume/µL	P265 Volume/µL	P266 Volume/µL	P267 Volume/µL	P268 Volume/µL
DNA (800 ng)	2.8	2.9	2.7	4.6	4.9
BSA (10x)	1	1	1	1	1
Buffer 4 (10x)	1	1	1	1	1
SpeI	0.5	0.5	0.5	-	-
XbaI	0.5	0.5	0.5	-	-
PstI	-	-	-	0.5	0.5
EcoRI	-	-	-	0.5	0.5
H2O	4.2	4.1	4.3	2.4	2.1
Total volume	10	10	10	10	10


Incubation time: 1 h; Incubation temperature: 37°
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , running time:5 minutes at 90 Volt and 40 minutes at 115 Volt
4µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)





Comment:pSB1C3_Rep68_ex clone 3 (P266) sent for sequencing with O162 (VF2) and O51 (VR2). pSB1C3_ZEGFR:1907 clone 1 (P267) sent for sequencing with O51 (VR2).

Hybridisation of the oligos for NLS and cloning NLS into pSB1C3

Investigator: Anissa

• Hybridisation: program: Origami 1
  Hybridisation was peformed according the standard protocol




• Digestion of pSB1C3_CFP
  

  components	Vector /µl
  DNA	6,62
  BSA (10X)	1,5
  Buffer 4	1,5
  Enzyme EcoRI	1
  Enzyme PstI	1
  H2O	3,38
  Endvolume	15




• Gel:After digestion, the sample was loaded on a 1% agarose-gel.




Digest pSB1C3_CFP




• Gelextraction: After the gel run, the vector backbone pSB1C3 was cut and purified according to the standard protocol




• Ligation: the original concentration of the hybridisation was 209,7 ng/µl, for the ligation a 1:10 dilution was made
  

  components	Volume /µl	Concentration/ ng/µl
  Insert (NLS)	6,79	2,097
  Vector (pSB1C3_cut)	1,21	8,3

  
  
• Transformation: Trafo into XL1 blue was performed according the standard protocol

Seeding HT1080 cells

Investigator: Kerstin, Adrian

• Seeding HT1080 cells for transduction with viral stocks from 18.08.
• We harvest four T75 flask (two of them were used three times (3 passages)) and seed them into eight 6er dishes (200.000 cells per well)

PCR and ligation for biobrick-production of pCerulean

Investigator: Christian L. , Stefan

DNA template: pEGFP_C1 (P230) C=367,4 ng/µl

PCR: (was performed following the standard protocol)

PCR program:

Comments: Errors in table, please correct!!! (TB)

Gelextraction of PCR product
Gelextraction was performed according to the standard protocol of the PCR product.

Comment: 0,4 g Agarose,50 ml TAE (0,8%), 3 µl GELRED , at 115 Volt, running time: 45 minutes

Digestion of PCR product and vector:

Purification of the PCR product with PCR purification was made according to the standard protocol.
Gelextraction of CFP
Gelextraction was performed according to the standard protocol.
>

Comment: 0.5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes

Ligation
The Ligation was performed as following:

• Vector Volume: 4,1 µl
• Insert Volume: 3,9 µl

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

Incubation for 45 minutes at room temperature
Transformation:
performed according to the standard protocol (XL1B). The cells were plated on a agar plate with Kanamycin.

Biobrick production of VP1_up

Investigator: Achim

DNA template: pKex_VP1 (P27) C=99,3 ng/µl

PCR: (was performed following the standard protocol)

PCR program:

Digestion of PCR product and vector:

Ligation with T4-Ligase and transformation with XL1B were carried out. Clones have to be picked tomorrow.

BioBrick assembly for pSB1C3_Affibody_Linker and pSB1C3_YFP_His-Tag

Investigators: Volker

The aim of this experiment is to assemble the affibodies with the the five linkers and the YFP with the His-Tag. All cloning is done according to RFC 25 and will be used to test the affibody if it really binds the EGF-receptor, for the N-terminal fusions and to supply it to other groups in the registry.

This experiment went wrong on Saturday 21.08. and was repeated on Sunday 22.08. and gave the following results. Results from the first experiment are not shown but can be senn in the written Labjournal.

Digestion

The following digestions were performed:

Incubation: 2 h

Preparative Agarose-Gel

1 g Agarose, 100 ml TAE (1%), 6 GELRED (3-6µl), at 120Volt, running time: 130 minutes

2 g Agarose, 100 ml TAE (2%), 6 GELRED (3-6µl), at 120 Volt, running time: 130 minutes

Sequencing results of pSB1C3_hTERT

Gel extraction

Ligation

96. labday 21.08.2010

Transfection of AAV293, Harvest viral particles, Transduction of HT1080 and Fluorescent microscopy

Investigators: Adrian, Kerstin

The Motivation
Measure the Transduction efficiency from 19.8

The Motivation
Transfection of AAV293 cells from 19.8 (ten 10cm² dishes with 2.000.000 cells each) with original standard protocoll!!! We want to try different 2xHBS Buffers (pH) and their influence on transfection efficiency!

The Plan

Requiered goods: 0,3 M CaCl₂ , 2x HBS-Buffer , autoclaved millipore water, falcons, eppis
Working steps:

1. Remove the three plasmids to be co-transfected (the recombinant pAAV expression plasmid or control plasmid, pAAV-RC, and pHelper) from storage at –20°C.
2. Pipet 10 µg of each of the three plasmid DNA solutions into an eppi.
3. Add 1ml of 0.3 M CaCl₂ and mix gently
4. Pipet 1ml of 2 × HBS into a 15-ml falcon.
5. pipet the DNA/CaCl₂ solution dropwise into the falcon (use the Pipetus).
6. Apply the DNA/CaCl₂/HBS suspension to the plate of cells in a dropwise fashion, swirling gently to distribute the DNA suspension evenly in the medium.
7. Return the tissue culture plate to the 37°C room for 6 hours.
8. At the end of the incubation period, remove the medium from the plate and wash cells with warm PBS, replace it with 10 ml of fresh DMEM growth medium.
9. Return the plate to the 37°C incubator for an additional 72 hours.

We try 5 different 2xHBS buffers:

• 1. Plate: 10µg of Rep/Cap, pHelper and GOI=mVenus; pH(2xHBS)= 7.06
• 2. Plate: 10µg of Rep/Cap, pHelper and GOI=mVenus; pH(2xHBS)= 7.08
• 3. Plate: 10µg of Rep/Cap, pHelper and GOI=mVenus; pH(2xHBS)= 7.10
• 4. Plate: 10µg of Rep/Cap, pHelper and GOI=mVenus; pH(2xHBS)= 7.12
• 5. Plate: 10µg of Rep/Cap, pHelper and GOI=mVenus; pH(2xHBS)= 7.14
• 6. Plate: 10µg of Rep/Cap, pHelper and GOI=TKGMK; pH(2xHBS)= 7.08
• 7. Plate: 10µg of Rep/Cap, pHelper and GOI=TKGMK; pH(2xHBS)= 7.10
• 8. Plate: 10µg of Rep/Cap, pHelper and GOI=mVenus; pH(2xHBS)= 7.14

The transfected cells were seeded at 19.8 (2.000.000 cells per 10 cm²)
Used Plasmids: Standart protocol: 10µg of each plasmid

• GOI: mVENUS => P262, conc:1148ng/µl; used amount: 8,71µl
• GOI: TKGMK => P82b, conc:1500ng/µl; used amount: 6,67µl
• pHELPER => P64, conc:503ng/µl; used amount: 20µl
• RepCap => 10.8: 1348ng/µl; used amount: 7,41µl




• Harvest viral paricles from 18.08.2010 and transduction of HT1080 cells following the standard protocol




• Analysis via Fluorescent microscopy (Transduction from 19.08.2010)
• Results:

Midi-Prep of pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1

Investigators: Chris W.

Comment: Midi-Preps of P 242, B200

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

Re-Trafo of pSB1C3_SDM_SspI_Bla14FM

Investigator: Stefan

Comment: Mini-Prep of pSB1C3_SDM_SspI_Bla14FM showed extremely low plasmid concentrations. It was proposed that this could correspond to using not the standard lab cells but rather XL-10 Gold cells for trafo. Therefore a re-trafo was performed using BL21 cells. 2 µl of each clone was added to BL21 cells and trafo was performed. Clones can be picked tonight (Volker)!

97. labday 22.08.2010

Test-digestion of pCerulean (P273,4,5), pSB1C3_VP1up (P279, 280), pSB1C3_NLS (P281,282), pAAV_RC_final (P283)

Investigator: Patrick
Digestions:

• 0,5 µl EcoRI, 0,5 µl Enzyme PstI, 1 µl BSA (10x), 1 µl Buffer 4 (10x), 1 µl pCerulean (P73), 6 µl H2O; total volume = 10 µl
• 0,5 µl EcoRI, 0,5 µl Enzyme PstI, 1 µl BSA (10x), 1 µl Buffer 4 (10x), 1 µl pCerulean (P74), 6 µl H2O; total volume = 10 µl
• 0,5 µl EcoRI, 0,5 µl Enzyme PstI, 1 µl BSA (10x), 1 µl Buffer 4 (10x), 1 µl pCerulean (P75), 6 µl H2O; total volume = 10 µl
• 1 µl NotI, 1 µl BSA (10x), 1 µl Buffer 4 (10x), 1 µl pSB1C3_VP1up (P279), 6 µl H2O; total volume = 10 µl
• 1 µl NotI, 1 µl BSA (10x), 1 µl Buffer 4 (10x), 1 µl pSB1C3_VP1up (P280), 6 µl H2O; total volume = 10 µl
• 0,5 µl EcoRI, 0,5 µl Enzyme PstI, 1 µl BSA (10x), 1 µl Bufer 4 (10x), 1 µl pSB1C3_NLS (P281) , 6 µl H2O; total volume = 10 µl
• 0,5 µl EcoRI, 0,5 µl Enzyme PstI, 1 µl BSA (10x), 1 µl Bufer 4 (10x), 1 µl pSB1C3_NLS (P282) , 6 µl H2O; total volume = 10 µl
• 0,5 µl Acc65I, 0,5 µl BspMI, 1 µl BSA (10x), 1 µl Buffer 3 (10x) , 1 µl pAAV_RC_final , 6 µl H2O; total volume = 10 µl

Incubation time: 37°C, 1h

Expected size of the fragments:

• pCerulean clone 1 : 771 & 3915 bp
• pCerulean clone 2 : 771 & 3915 bp
• pCerulean clone 3 : 771 & 3915 bp
• pSB1C3_VP1up clone 1 : 468 & 2030 bp
• pSB1C3_VP1up clone 2 : 468 & 2030 bp
• pSB1C3_NLS clone 1 : 78 & 2031 bp
• pSB1C3_NLS clone 2 : 78 & 2031 bp
• pAAV_RC_final : 615 & 6720 bp

pCerulean clone 1 (P273), pSB1C3_VP1up clone 2 (P280), pSB1C3_NLS clone 2 (P282) and pAAV_RC_final (P283) were sent for sequencing although the results don't look very promising.
Labelling:

• pCerulean clone 1 (P273) : iGEM2-CMV-F
• pSB1C3_VP1up clone 2 (P280) : iGEM1-VF2
• pSB1C3_NLS clone 2 (P282) : iGEM276-O51_VR2 and iGEM3-O51_VR2 (was ordered twice due to an ordering mistake)
• pAAV_RC_final (P283) : iGEM4-SK
• pAAV_RC_final (P283) : iGEM4-VP1 pKEX rev
• pAAV_RC_final (P283) : iGEM4-Cap2800for
• pAAV_RC_final (P283) : iGEM4-Cap2800rev
• pAAV_RC_final (P283) : iGEM4-Cap3500for

Mini-Prep of pCerulean

Investigator: Stefan

Glycerol stocks were prepared:
• B225 = pCerulean clone1
• B226 = pCerulean clone2
• B227 = pCerulean clone3

Mini-Prep following the standard protocol

• P273 = pCerulean clone1 c= 419,52 ng/µl
• P274 = pCerulean clone2 c= 383,37 ng/µl
• P275 = pCerulean clone3 c= 382,72 ng/µl

Test digestion will be performed tomorrow.

98. labday 23.08.2010

Primer design: Sequencing primer for BioBrick Assembly of vectorplasmid and phTERT qPCR primer

Investigator: Bea

Comment: Two goals will be accomplished with this primers.

1. The first primer pair (left picture) serve as sequencing primers for the pSB1C3 vector plasmid. Problem was that the designed VR-2/VF-2 primers cannot be used for sequencing because the ITRs flank several assembled parts, mostly CMV, beta globin, GOI and hGH. These primers were designed to bind in the flanked region, directly after/before left and right ITR repsectivel and can therfore be used to sequence most of the flanked sequence inbetween the ITRs.
2. The second primer pair (right picture) will be used for performing the qPCR with th econtruct containing the phTERT promoter. The qPCR primers used for the other qRT-PCR cannot be used because these two primers anneal in the CMV region. The new designed primers bind in the phTERT region and can therefore bee used for determinining the infectious titers.

Primer design. Ordered at Sigma Aldrich

Mini-Prep of pSB1C3_CFP_middlelinker and pSB1C3_ZEGFR:1907 / testdigestion of pSB1C3_CFP_middlelinker and pSB1C3_lITR_pHTERT

Investigator: Jessica

Mini-Prep following the standard protocoll

• P276 PSB1C3_CFP_middlelinker clone1 c=207,7 ng/µl
• P277 PSB1C3_CFP_middlelinker clone2 c=203,0 ng/µl
• P278 PSB1C3_CFP_middlelinker clone3 c=227,9 ng/µl
• P279 pSB1C3_VP1up clone1 c=183,0 ng/µl
• P280 pSB1C3_VP1up clone2 c=178,1 ng/µl
• P281 pSB1C3_NLS clone1 c=155,7 ng/µl
• P282 pSB1C3_NLS clone2 c=142,6 ng/µl
• P283 pAAV_RC_final c=147,7 ng/µl
• P284 pSB1C3_ZEGFR:1907 clone 1 c=146,5 ng/µl
• P285 pSB1C3_ZEGFR:1907 clone 1 c=161,9 ng/µl
  

pSB1C3_Zegfr:1907 wurde bereits von Stefan geprept... was hast du hier gemacht Jessy? Retrafo vom Glycerolstock???

Test digestion:
• Perfomed with 10 µL of total volume

• incubation time: 50'
• incubation temparatur: 37°C

• 5µl new ladder mix

Sequencing:

• Plasmid used: P276: pSB1C3_CFP_middlelinker
• Primer used: VR-2
• Tube name: JG 276

Continuation of BioBrick assembly for pSB1C3_Affibody_Linker

Investigator: Anna

Comment: Transformation was done of pSB1C3_Affibody with Short-Linker, Middle-Linker, Long-Linker, SEG-Linker and GSAT-Linker. Unfortunately, the digestion of pSB1C3_YFP_His-Tag didn't work (see 20.08).

Transformation was done following the standard protocol using XL1 blue cells.

To do: Repetition of BioBrick assembly for pSB1C3_YFP_His-Tag.

Cloning of pSB1C3_ß-globin_YFP and pSB1C3_hGH_rITR

Investigator: Anna

Comment: pSB1C3_ß-globin_mVenus (p178) was digested with SpeI and PstI and pSB1C3_hGH_rITR (p186) was digested with XbaI and PstI .

Vector:

• pSB1C3_ß-globin_YFP (p178): c= 237,2 ng/µl
  

Insert:

• pSB1C3_hGH_rITR (p186): c= 105,2 ng/µl
  

• Gelextraction of pSB1C3_RFC25_CFP:

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time:55
Marker: GeneRuler ladder mix (Fermentas)

• c(pSB1C3_ß-globin_YFP)= 16.9 ng/µl; size: 3293 bp
  
• c(hGH_rITR)= 7,5 ng/µl; size: 657 bp

• Ligation of PCR products and vector:

For the Ligation 1µl T4 buffer (2x) and 1µl T4 ligase were used. Incubation time: 30 min

• Transformation:

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

To do: Picking clones of pSB1C3_ß-globin_mVenus_hGH_rITR.

Repetition: Quickchange site-directed mutagenesis of pSB1C3_SDM_SspI_Bla14FM

Investigator: Stefan

Comment: To delete a PvuII restriction site in the CAT marker, a SDM will be performed.

• PCR reaction:

• PCR program:

SDM was performed according to protocol.

Transformation was performed according to protocol using XL1b cells.

FACS analysis, Seeding HT1080 and Hek293 cells

Investigator: Kerstin, Adrian

• Seeding HEK293 cells (100.000, 200.000, 300.000, 400.000, 500.000, 600.000 and 700.000 cells per dish)for transfection

• FACS analysis of transduction from 21.08.2010:

• Seeding HT1080 cells for transduction with viral stocks from 23.08.2010:
  • 3x 6er wells(Transductionplan 23.08.2010):

Miniprep of pSBC13_Bla

Investigator: Chris L.

Plasmid Mini-Prep

Gycerol stock was prepared and labeled for pSB1C3_Bla clone 1.1 (B236), 1.2 (B237), 2.1 (B238) and 2.2 (B239).B167.

Miniprep was performed according to protocol.

Samples were labeled:

• pSB1C3_Bla clone 1.1 = P286
• pSB1C3_Bla clone 1.2 = P287
• pSB1C3_Bla clone 2.1 = P288
• pSB1C3_Bla clone 2.2 = P289

Concentrations:

• pSB1C3_Bla clone 1.1 (P286): 95.24 ng/µl
• pSB1C3_Bla clone 1.2 (P287): 84.92 ng/µl
• pSB1C3_Bla clone 2.1 (P288): 43.66 ng/µl
• pSB1C3_Bla clone 2.2 (P289): 43.95 ng/µl

Test digestion: pSB1C3_Bla 1.1, 1.2, 2.1 and 2.2 were digested with NotI. pSB1C3_Bla 1.1, 1.2, 2.1 and 2.2 were digested with SspI and SalI. pSB1C3_Bla 1.1, 1.2, 2.1 and 2.2 were digested with BamHI and PvuII.

Incubation time: 1 h, Incubation temperature: 37°
2 Gels were made:
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , running time: 15 minutes at 90 Volt and 30 minutes at 120 Volt
2µl loading dye (6x) for the sample, Marker: 5 µl GeneRuler ladder mix (Fermentas)

• Marker: GeneRuler ladder mix

• Marker: GeneRuler ladder mix

Comments: Strange results, seems like the test digestion didn't work out. Repitition of test digestion tomorrow.

99. labday 24.08.2010

Sequence analysis of pSB1C3_CFP_middlelinker

Investigator: Jessica

• pSB1C3_CFP_middlelinker is ready

Harvest viral particles, Transduction of HT1080

Investigator: Kerstin

• Harvest viral particles following the standard protocol

• Transduction of three 6-well plates:

Plate 1 YFP: 150.000 cells per well

Plate 2 YFP: 150.000 cells per well

Plate 3 YFP: 200.000 cells per well

• (1)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,06
• (2)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,08
• (3)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,10
• (4)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,12
• (5)= 10µg RC, 10µg pHelper, 10µg GOI (YFP); pH(2xHBS)=7,14
• (9)= 10µg RC, 10µg pHelper, 3,3µg GOI (eGFP)

Mini-prep of SDM NgoMIV CD

Investigator: Kira
Motivation: Harvest DNA for further analysis, e.g. test digestion

Mini-prep was performed on 3 colonies.

c(clone1) = 358, 05 ng/ul c(clone2) = 352, 10 ng/ul c(clone 3) = 376, 53 ng/ul

Sent for sequencing: pSB1C3_6xHis

Investigator: Jessica

• Plasmid: P84 c= 107,8 ng/µl
• Primer: VR2
• tube number: JG84

Subcloning Cap into pAAV_RC_ins-rep

Investigator: Stefan

Comment: Sequencing of pAAV_RC_final showed that Cap was not inserted succsessfully. Therefore it has to be repeated.

Digestion of vector and insert:

• Insert: pMA_RepCap Vector_SDM_InsPvuII clone 1 (P211)
• Vector: pAAV_RC_ins-rep clone1 (P250)
  

A two-step digestion was performed:

1st step

2nd step

Samples were loaded on a 0,8% agarose gel an run at 115V for about 60 minutes.

pAAV RC final

Gelextraction:

Gelex was performed according to protocol exept for elution was done with 60 µl instead of 20 µl.

• c(P211)= 1,14 ng/µl
• c(P250)= 5,60 ng/µl

T4 Ligation:
T4 ligation was performed according to protocol. used DNA amounts:

• v(P211)= 2,56 µl
• v(P250)= 5,44 µl

Transformation:

Trafo was performed according to protocol using XL1b cells.

Test digestion of pCerulean (p273)

Investigator: Anissa

Comments:Sequencing of pCerulean showed strange results, because the qualtity of sequenzing was not good. For working further with pCerulean, we started with a test digestion, to be sure, everything works.
Because the sequencing showed no AgeI, we cut one time with AgeI and EcoRI, the other time with NotI.

Comments: Results of digestion seem to be all right

Cloning of VP1up into PCerulean

Investigator: Anissa
VP1up was cut out of pSB1C3 and into pCerulean

• Digestion:
  
  

  Components	Vector/µL	Insert/µL
  DNA	3,7	8,4
  BSA (10x)	1,5	2
  Buffer 4 (10x)	1,5	2
  Enzyme 1 EcoRI	1	1
  Enzyme 2 PstI	1	1
  H2O	6,3	5,6
  Total volume	15	20

  
  

• Gel:
  A 1% agarose-gel was made, after 30 minutes samples were cut
  
  
• Gelextraction was performed according the standardprotocol
• Ligation:

  Components	used volume for T4 ligation/µL	concentration /ng/µl
  Vector	5,15	27,7
  Insert	2,85	15,4

  In addition 1µl T4-ligase and 1µl T4-ligase-buffer were added

• Transformation: was performes into Xl1 blue according the standard-protocol

Sequence analysis of sequencing reads prepared yesterday (23.08.2010)

Investigator: Bea

GENERAL COMMENT: pSB1C3 {NLS and VP1up} were sent for sequencing. Aswell as the pAAV_RC_final which contains all four mutations in the Rep-Cap gene and the integrated and synthesiezd rep and cap gene and the plasmid pCerulean whith the CMV promoter and sv40 polyadenylation site.

Comments: Sequence analysis of pAAV_RC_final containing the subcloned "rep" (ordered) and "Cap (ordered) plus the four mutations to delete the restriction sites PstI, BamHi and SalI.

• Primer used:
• VP1 primer for pKex
• GATC_std_SK
• Primer Cap 2800 rev
• Primer Cap 2800 for
• Primer Cap 3500 for
• Plasmid sequenced: P283
• Sequence sample: iGEM4
• Stored in Geneious-folder: RepCap insert ordered

pAAV RC final

Sequencing of the mutation ok. Rep integration worked. Cap integration needs to be repeated.

Next steps: Repetition of the integration of the synthesized cap gene performed by Stefan (see topic).

Comments: Sequence analysis of pSB1C3_NLS

• Primer used: VR-2
• Plasmid sequenced: P282
• Sequence sample: iGEM3_O51_VR-2
• Stored in folder: pSB1C3_NLS

pSB1C3_NLS

Insertion of the nuclear localisation signal (NLS) into the pSB1C3_NLS as one step in the N-terminal insertion of targeting molecules. Sequencing results look good. prefix and suffix ok!

Next steps: Fuse NLS to targeting molecule.

Comments: Sequence analysis of pSB1C3_VP1up

• Primer used: VF-2
• Plasmid sequenced: P280
• Sequence sample: iGEM1_VF-2
• Stored in folder: N-Terminal targeting --> pSB1C3_VP1up

pSB1C3_VP1up

Prefix ok, but annotation is wrong. VP1up should start with: GC!! Suffix ok! The additional EcoRI before the prefix standard can be due to the bad sequence read quality.

Next steps: Clone VP1up into pCerulean in order to obtain the plasmid with a CMV promoter and the sv40 polyadenylation site.

Comments: Sequence analysis of pCerulean

• Primer used: GATC_CMV-F
• Plasmid sequenced: P273
• Sequence sample: iGEM2_CMV-F
• Stored in folder: N-Terminal targeting --> pCerulean

pCerulean

It seem that the suffix is not correct which means that AgeI, PstI and NotI is missing. This can be due to the bad sequence quality. Prefix is ok!

Next steps: For verification a test digestion with the missing restriction sites will be perforemd and another round of sequencing will be conducted.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Test digestion of SDM NgoMIV CD

Investigator: Kira
Motivation: In order to check if site directed mutagenesis was successfull, test digestion was performed with 3 clones, which were picked yesterday as well as with 'original' DNA, which still contains all the restriction sites

Bitte schneid mich aus !

According to the gel, NgoMIV SDM was successful in clones 1 and 2. Clone 3 well reveales 2 bands of unknown origin. Samples 1 and 3 were sent for sequencing.

Picking clones of pSB1C3_Affibody_linker and pSB1C3_ß-Globin_YFP_hGH_rITR

Investigator: Anna

Comments: A 1:1000 dilution of pSB1C3_Affibody_GSAT-Linker was prepared.
To do: Mini-Prep of pSB1C3_Affibody_(Short/Middle/Long and SEG-Linker) and pSB1C3_ß-Globin_YFP_hGH_rITR. Picking clones of pSB1C3_Affibody_GSAT-Linker.

Test digestion of pSB1C3_SDM_SspI_Bla14FM (P222), pSB1C3_BLA (P286, P288)

Investigator: Achim

CommentsNew test digestion of two SDM-attempts, this time we also digested the original vector containing the PvuII restriction site to see differences.

• The original vector is being cut as expected: the uncut vector can be seen in different conformations, the linearized vector shows one distinct band and the digestions cutting out BLA show a band at ~900 bp. Our SDM attempts show inconsistent results, not only does the Pvu site still seem to be in the backbone, there are no more proper insert bands visible either. We therefore conclude that either the DpnI digestion or the dilution of the transformation must have gone wrong (differing plasmids...). Tomorow we'll try a new cloning approach using an old vector with deleted Pvu restriction site and MscI & XbaI digestion. In case this also fails we also yet have to prep and test digest the repetition of the mutagenesis which was carried out by Stefan.

pSB1C3_VP1up

Prefix ok, but annotation is wrong. VP1up should start with: GC!! Suffix ok! The additional EcoRI before the prefix standard can be due to the bad sequence read quality.

Next steps: Clone VP1up into pCerulean in order to obtain the plasmid with a CMV promoter and the sv40 polyadenylation site.

Comments: Sequence analysis of pCerulean

• Primer used: GATC_CMV-F
• Plasmid sequenced: P273
• Sequence sample: iGEM2_CMV-F
• Stored in folder: N-Terminal targeting --> pCerulean

pCerulean

It seem that the suffix is not correct which means that AgeI, PstI and NotI is missing. This can be due to the bad sequence quality. Prefix is ok!

Next steps: For verification a test digestion with the missing restriction sites will be perforemd and another round of sequencing will be conducted.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Test digestion of SDM NgoMIV CD

Investigator: Kira
Motivation: In order to check if site directed mutagenesis was successfull, test digestion was performed with 3 clones, which were picked yesterday as well as with 'original' DNA, which still contains all the restriction sites

Bitte schneid mich aus !

According to the gel, NgoMIV SDM was successful in clones 1 and 2. Clone 3 well reveales 2 bands of unknown origin. Samples 1 and 3 were sent for sequencing.

Picking clones of pSB1C3_Affibody_linker and pSB1C3_ß-Globin_YFP_hGH_rITR

Investigator: Anna

Comments: A 1:1000 dilution of pSB1C3_Affibody_GSAT-Linker was prepared.
To do: Mini-Prep of pSB1C3_Affibody_(Short/Middle/Long and SEG-Linker) and pSB1C3_ß-Globin_YFP_hGH_rITR. Picking clones of pSB1C3_Affibody_GSAT-Linker.

Test digestion of pSB1C3_SDM_SspI_Bla14FM (P222), pSB1C3_BLA (P286, P288)

Investigator: Achim

CommentsNew test digestion of two SDM-attempts, this time we also digested the original vector containing the PvuII restriction site to see differences.

• The original vector is being cut as expected: the uncut vector can be seen in different conformations, the linearized vector shows one distinct band and the digestions cutting out BLA show a band at ~900 bp. Our SDM attempts show inconsistent results, not only does the Pvu site still seem to be in the backbone, there are no more proper insert bands visible either. We therefore conclude that either the DpnI digestion or the dilution of the transformation must have gone wrong (differing plasmids...). Tomorow we'll try a new cloning approach using an old vector with deleted Pvu restriction site and MscI & XbaI digestion. In case this also fails we also yet have to prep and test digest the repetition of the mutagenesis which was carried out by Stefan.

100. labday 25.08.2010

Cloning of pSB1C3_Rep40, pSB1C3_Rep68 and pMA_RC_insert

Investigator: Chris L.

• buffer used: 2; Restriction-enzymes used: Enzyme 1: HindIII ; Enzyme 2: BstEII

Plasmids:

• pSB1C3_Rep40 P231
• pSB1C3_Rep68 P266

Insert:

• pMA_RC_insert P190

• Incubation: 90 minutes; 37°C with HindIII
  
• Incubation: 90 minutes; 60°C with BstEII
  

Agarosegel
0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, 5 min at 90 Volt, 40 min at 115 Volt

Concentrations measured via NanoDrop:

• pSB1C3_Rep40: 81.97 ng/µl
• pSB1C3_Rep68: 48,68 ng/µl
• RC_Insert: 3,60 ng/µl

T4 Ligation of pSB1C3_Rep40 with RC_Insert
Volume insert: 6,54 µl
Volume vector: 1,46 µl

T4 Ligation of pSB1C3_Rep68 with RC_Insert
Volume insert: 5,46 µl
Volume vector: 2,54 µl

Trafo was prepared with XL1blue and Cm

Picking clones of pAAV_RC-ins_rep_cap and pCeruleanVP1up

Investigator: Chris L.

To do: Mini-Prep of pAAV_RC-ins_rep_cap and pCeruleanVP1up.

Cloning of pSB1C3_6xHis with middlelinker and pSB1C3_6xHis with pGA14_mVenus_YFP

Investigator: Jessica

• buffer used: 4; Restriction-enzymes used: Enzyme 1: AgeI ; Enzyme 2: PstI ; Enzyme3: NgoMIV

• Plasmids:
• pSB1C3_6xHis P84
• pGA14_middle linker P65
• GA14_mVenus_YFP P60

• Incubation:110 minutes; 37°C
  

Agarosegel
0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, at 120 Volt

concentrations measured via NanoDrop:

• SB1C3_6xHis: 1,7 ng/µl
• middle linker: 3,8 ng/µl
• mVenus_YFP: 3,2 ng/µl

T4 Ligation pSB1C3_6xHis_middlelinker
Volume insert: 0,25 µl
Volume vector: 7,75 µl

T4 Ligation pSB1C3_6xHis_mVenus_YFP
Volume insert: 2,64 µl
Volume vector: 5,36 µl

Trafo was prepared with XL1blue and Cm

Sequence analysis of pSB1C3_6xHis

Investigator: Jessica
P84 was sequenced for continuation of working with it

• pSB1C3_6xHis P84 is ready to use, but is empty. a new tube will be made tomorrow

Inoculation of pGA14_middlelinker and pSB1C3_6xHis clone1

Investigator: Jessica

• both tubes (P65 and P84)are empty --> inoculation with glycerolstocks
  • B48 pGA14_middlelinker was inoculated with 10ml DYT and 10µl Amp
  • B64 pSB1C3_6xHis clone1 was inoculated with 10ml DYT and 10µl Cm

Cloning of pSB1C3_SspI_BLA and pSB1C3_CFP_PvuII

Investigator: Achim

Comment:I ligated a sequence from pSB1C3_CFP_PvuII missing the PvuII restriction site into pSB1C3_SspI_BLA to obtain a pSB1C3 vector without PvuII in the cat marker.

Vector:

• pSB1C3_SspI_BLA (p222): c= 308,32 ng/µl
  

Insert:

• pSB1C3_CFP_PvuII (p129): c= 264,86 ng/µl
  

• Gelextraction:

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time:45
Marker: GeneRuler ladder mix (Fermentas)

• c(Insert)= 15,66 ng/µl; size: 730 bp
  
• c(Vector)= 14,42 ng/µl; size: 2200 bp

• Ligation of PCR products and vector:

For the Ligation 1µl T4 buffer (2x) and 1µl T4 ligase were used. Incubation time: 60 min due to blunt end ligation

• Transformation:

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

Comment: Two clones were picked, but because we already obtained our plasmid via SDM in the meantime, no prep was performed. Glycerol stocks of the two clones were created just in case, B264 and B265.

Mini-Preps and test digestion of pSB1C3_Affibody_Middle-Linker, pSB1C3_Affibody_Short-Linker, pSB1C3_Affibody_SEG-Linker, pSB1C3_Affibody_Long-Linker and pSB1C3_betaglobin_mVenus_hGH_rITR

Investigator: Stefan

Glycerol stocks were prepared:
• B240 = pSB1C3_Affibody_Middle-Linker clone1
• B241 = pSB1C3_Affibody_Middle-Linker clone2




• B242 = pSB1C3_Affibody_Short-Linker clone 1
• B243 = pSB1C3_Affibody_Short-Linker clone 2




• B246 = pSB1C3_Affibody_SEG-Linker clone1
• B247 = pSB1C3_Affibody_SEG-Linker clone2




• B248 = pSB1C3_Affibody_Long-Linker clone 1
• B249 = pSB1C3_Affibody_Long-Linker clone 2




• B244 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 1
• B245 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 2





Mini-Prep following the standard protocol

• P290 = pSB1C3_Affibody_Middle-Linker clone1 c= 227,36 ng/µl
• P291 = pSB1C3_Affibody_Middle-Linker clone2 c= 219,05 ng/µl




• P292 = pSB1C3_Affibody_Short-Linker clone 1 c= 196,63 ng/µl
• P293 = pSB1C3_Affibody_Short-Linker clone 2 c= 224,76 ng/µl




• P296 = pSB1C3_Affibody_SEG-Linker clone1 c= 218,68 ng/µl
• P297 = pSB1C3_Affibody_SEG-Linker clone2 c= 229,66 ng/µl




• P298 = pSB1C3_Affibody_Long-Linker clone 1 c= 229,73 ng/µl
• P299 = pSB1C3_Affibody_Long-Linker clone 2 c= 202,05 ng/µl




• P294 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 1 c= 425,14 ng/µl
• P295 = pSB1C3_betaglobin_mVenus_hGH_rITR clone 2 c= 328,28 ng/µl

Test digestion

• Restriction-enzymes used: for P290-P293 and P296-P299: NotI ; for P294-P295: NgoMIV and PstI

comment: The same amount of ingredients were used for P290-P293 and P296-P299, therefore these approaches will be merged into the chart. The same goes for P294-P295.

Incubation time: 70 minutes; Incubation temperature: 37°
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , running time:5 minutes at 90 Volt and 50 minutes at 115 Volt
2µl loading dye (6x) for the sample, Marker: GeneRuler ladder mix (Fermentas)

Comment: Sizes of fragments look like expected. Clone 1 of each plasmid will be sent for sequencing tomorrow.

Sequenzing evaluation of SDM NgoMIV

Investigator: Kira

2 samples have been sent for sequencing yesterday. According to the data, both samples do not contain any NgoMIV restriction site anymore.

101. labday 26.08.2010

Mini-Preps of pGA14_middle linker and pSB1C3_6xHis clone 1

Investigator: Jessica

Glycerol stocks were not prepared because I have inoculated from glycerol stocks
Mini-Prep following the standard protocol
• P301 = pGA14_middle linker (= P65)
  c=305,7




• P300 = pSB1C3_6xHis clone 1 (= P84)
  c=177,5

Mini-Preps and test digestion of pAAV_RC-ins_rep_cap, pCeruleanVP1up and pAAV_RC_ins-rep-cap.

Investigator: Chris L.

Glycerol stocks were prepared:
• B250 = pCerulean_VP1up clone1
• B251 = pCerulean_VP1up clone2
• B252 = pCerulean_VP1up clone3
• B253 = pCerulean_VP1up clone4
• B254 = pSB1C3_VCK_Bla clone1
• B255 = pSB1C3_VCK_Bla clone2
• B256 = pSB1C3_VCK_Bla clone3
• B257 = pSB1C3_VCK_Bla clone4
• B262 = pAAV_RC_ins-rep-cap clone1
• B263 = pAAV_RC_ins-rep-cap clone2


Mini-Prep following the standard protocol

• P302 = pCerulean_VP1up clone1
  c=410,70 ng/µl
• P303 = pCerulean_VP1up clone2
  c=387,78 ng/µl
• P304 = pCerulean_VP1up clone3
  c=321,04 ng/µl
• P305 = pCerulean_VP1up clone4
  c=350,34 ng/µl
• P306 = pSB1C3_VCK_Bla clone1
  c=82,53 ng/µl
• P307 = pSB1C3_VCK_Bla clone2
  c=92,70 ng/µl
• P308 = pSB1C3_VCK_Bla clone3
  c=82,22 ng/µl
• P309 = pSB1C3_VCK_Bla clone4
  c=105,34 ng/µl
• P314 = pAAV_RC_ins-rep-cap clone1
  c=517,04 ng/µl
• P315 = pAAV_RC_ins-rep-cap clone2
  c=444,33 ng/µl

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: 50 minutes

pSB1C3_VCK_Bla: Test digestion looks like expected. Clone 4 was sent for sequencing.
pCerulean_VP1up: Gel looks well as well. clone 2 was sent for sequencing.
pAAV_RC_ins-rep-cap: Test digestion looks strange. Just one big band with more than 10000 bp and one very small with 150 bp. Maybe the restriction enzymes didn´t cut. (@Christian: please insert picture)

102. labday 27.08.2010

Sequence analysis of pSB1C3_Bla_final (P309)and pCerulean_VP1up (P303)

Investigator: Bea

GENERAL COMMENT: pSB1C3_Bba_final (P309, SB_9) and pCerulean_Vp1up (P303; SB_10) were sent for sequencing. In the case of SB1C3_Bba_final, which means that the whole vector was assembled from the buttom,the purpose was to test the designed primers. In the other case we wanted to verify the insertion of Vp1up inot pCerulean.

Comments: Sequence analysis of pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR.

• Primer used:
• pSB1C3_Bba_seq_for
• pSB1C3_Bba_seq_rev
• Plasmid sequenced: P??
• Sequence sample: ??
• Stored in Geneious-folder: BioBricks --> final parts

pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR

Results look good. The forward primer worked, still waiting for the results of the reverse primer. The scar inbetween the CMV promoter and the beat globin intron corresponds to the expected result.

Next steps: Wwait until sequencing results of the reverse primer can be confirmed aswell. If that will not be the case, order new sequensing primer.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Comments: Sequence analysis of pCerulean_Vp1up.

• Primer used: CMV-F
• Plasmid sequenced: P303
• Sequence sample: ??
• Stored in Geneious-folder: N-terminal Targeting --> pCerulean_Vp1up

pCerulean_Vp1up

Results are good. Insertion of the Vp1up region can be confirmed.

Next steps: Fuse pCerulean_Vp1up to the NLS.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Hybridisation and cloning of loop insertions

Investigator: Achim, Volker

We hybridized the oligos for the different loop insertions and cloned them into the pSB1C3_BLA backbone.

Hybridisation

• 4 Inserts contained overlapping ends and had to be filled up using Klenov fragments. We therefore added Klenow buffer and dNTPs to those samples.

• Hybridisation was carried out according to standard protocoll
• Klenov fill-in reaction:
  • Added 1 µl of NEB Klenow fragment to samples 11,12,13,14
  • incubated for 1h @ 37°C

Digestion of pSB1C3_BLA vector and Samples 11-14

• We digested the standard vector with the 453 (Ssp/Sal) and the 587 (Bam/Pvu) standard. The samples that were filled in were also digested to create sticky ends.

Gel Extraction

Ligation

Cloning of pCerulean_VP1up_NLS

Investigator: Anissa

Comment:Cloning did not work, because no NLS-band could be seen in the gel... Will be repeated on monday in two new approaches. One time VP1up will be cloned into pSB1C3_NLS and recloned as a fusion-product into pCerulean. Another time the oligos of NLS will be hybridisized, cut with rescriction enzymes and purificated with the Qiaex II kit.Then the NLS will be ligated into the pCerulean_VP1up

• Digestion:

• Gel: that's only the picture of the 2% agarose gel for seperating the NLS. But no band could be seen.

DARPin E_01

Investigator: Bea

General overview of DARPins:

Natural protein ankyrin repeat molecules are motifs which can be found in proteins. These motifs are mediating protein-protein interactions. This suggests that ankyrin repeat (AR) proteins can be used for designing binding molecules. In Kohl et al. (2003) and Binz et al. (2003) the authors designed a structural framework with fixed consensus regions and randomized positions of interacting residues.

The repetitive nature of the ankyrin proteins allows modifications in their variable and modular binding surface. Therefore consensus sequences of natural ankyrin proteins were used to design novel and stable scaffolds for binding proteins. Designed Ankyrin Proteins (DARPins) are well expressed, monomeric in solution, thermodynamically stable and have the ability to fold fast. In the publication of Steiner et al. (2008) screening libraries were created by useing the signal recognition particle (SRP) translocation pathway for phage display. The proteins containing the appropriate translocation signal sequence are efficiently displayed on filamentous phage particles. Screening for DARPins binding to specific target proteins like EGF-R (Erb1) were performed by phage ELISAs. The extracellular domain I-III of the receptor ErbB1 was fused to the Fc part of human IgG1 and used for selection. The previously described combinatorial N3C library was used as a template for SRP selection. After phage ELISA ans sequencing have been performed only one clone (E_01) could be selected with high-affinity binding characteristics. For broaden the diversity of ErbB1 DARPins three more clones were selected by epitope masking (E_67, E_68 and E_69). Binding and epitope localization experiments showed that all selected clones recognize a competing epitope as the dominant binder E_01, except of E_69 which recognizes an epitope not competing with E_01. The low diversity obtained for DARPins against ErbB1 suggests that the binding interfaces are well suited for dominant selection. Additionally the selected clones showed no cross-reactivity with other ErbB-family receptors (Figure 2C).

The dominant DARPpin E_01 has very high affinities to the target protein ErbB1 (Table 3) and can be used as a potential targeting molecule four our approach in fusing the DARPin to the N-terminal VP proteins.

Overview of DARPinE_01 used in our approach:

The designed ankyrin repeat protein used as a potential targeting molecule consists of three internal capping repeats and the C-and N-terminal capping repeats. Each internal repeat module comprises of one beta-turn and two hydrophobic alpha-helices. The potential interaction residues are located in the beta-turn the first alpha-helices of the AR-proteins. The complete nucleotide and corresponding amino acid sequence can be found in the appendix.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Update VP1 insertion

Investigator: Anissa, Bea

Done:

• pSB1C3_VP1up was succesfully created by Achim. For details see labday from 2010-22-08.
• pCerulean_VP1up was suceesfully created by Anissa. pCerulean was sequenced and the VP1up which contains the upstream 137 aa´s of the VP1 protein could be successfully cloned into the plasmid backbone pCerulean (obtained by performeing a PCR).
• pSB1C3_NLSHybridization of the nuclear localization sequence (NLS) was successfully incorporated into the pSB1C3_CFP.
• Today, Anissa tried to fuse the NLS to the VP1up sequence. Cutting the pSB1C3_NLS with NgomIV and PstI leads to a 45 nt fragment which normally can be resoluted in a 2% agarose gel. But no fragment could be detected in the gel (for further details see: Cloning of pCerulean_VP1up_NLS).
  On monday another approach will be performed: The NLS oligos will be hybridized and digested with NgomIV and SpeI. With the Qiaex II Kit (protocol for desalting and concentrating DNA solutions),the hybrifized oligos will be purified. Parallel, another digestion approach will be conducted.

Overview

Next steps:

• We obtain pSB1C3_VP23_HSPG_ko by conducting a PCR with the pAAV_RC_final (which contains the integrated "cap".
• Fuse the obtained construct pSB1C3_Targeting Molcule to the pSB1C3_VP23_HSPG_ko.
• Affibody Z_EGFR:1907
• DARPin E_01
• 6xHis Tag
• CFP
• Fuse pSB1C3_VP23_HSPG_ko_Targeting molecule to the construct pCerulean_VP1up_NLS.
• Finally, for providing the possibilty to express obtained construct in trans: perform a site-directed mutagenesis with the pAAV_RC_final to remove the startcodon of VP1.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Sequence analysis of pSB1C3_BLA_final (P309)

Investigator: Bea

GENERAL COMMENT: pSB1C3_BLA_final (P309) was sent for sequencing. the beta lactamse was inserted and two site-directed mutagenesis have been performed in order to delete the loop insertion restriction enzymes in the CAT marker.

Comments: Sequence analysis of pSB1C3_BLA_final containing the two deleted restriction sites (SspI and PvuII)in the CAT marker.

• Primer used:
• pQE-RP
• VF-2
• Plasmid sequenced: P309
• Sequence sample: SB_9
• Stored in Geneious-folder: pSB1C3_BLA

pSB1C3_BLA_final

The two performed site-directed mutagenesis performed at the backbone of the pSB1C3 containing the beta-lactamase can be confrimed as it can be seen in the two pictures above. Additionally the BLA sequence was sequenced aswell as the insertion of the BLA which can be confirmed with no mutations.

Next steps: This new plasmid can be used for further experiments. Clone the PCR prdouct of the pAAV_RC into the pSB1C3 and subclone the loop insertion motifs into the vector.

[http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/Laborjournal top of page]

Mini-Preps and test digestion of pSB1C3_6xHis_middlelinker and pSB1C3_6xHis_mVenus

Investigator: Stefan

Glycerol stocks were prepared:
• B258 = pSB1C3_6xHis_middlelinker clone1
• B259 = pSB1C3_6xHis_middlelinker clone2




• B260 = pSB1C3_6xHis_mVenus clone 1
• B261 = pSB1C3_6xHis_mVenus clone 2




Mini-Prep following the standard protocol

• P310 = pSB1C3_6xHis_middlelinker clone1
  c= 185,31 ng/µl
• P311 = pSB1C3_6xHis_middlelinker clone2
  c= 178,87 ng/µl




• P312 = pSB1C3_6xHis_mVenus clone 1
  c= 247,62 ng/µl
• P313 = pSB1C3_6xHis_mVenus clone 2
  c= 268,98 ng/µl

Test digestion

• Restriction-enzymes used: EcoRI and PstI

Incubation time: 90 minutes; Incubation temperature: 37°
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , running time: 55 minutes at 120 Volt

Comment: Test digestion looks good. Clones 1 of each plasmid (P310 and p312) were sent in for sequencing.

Repetition of test digestion of pAAV_RC_ins-rep-cap, pAAV_RC_ins-rep, pSB1C3_Rep40_RC_Insert and pSB1C3_Rep68_RC_Insert.

Investigator: Chris L.

• P250 = pAAV_RC_ins-rep clone1
  c=467,50 ng/µl
• P314 = pAAV_RC_ins-rep-cap clone1
  c=517,04 ng/µl
• P315 = pAAV_RC_ins-rep-cap clone2
  c=444,33 ng/µl




• P316 = pSB1C3_Rep40_ins clone 1
  c=302,22 ng/µl
• P317 = pSB1C3_Rep40_ins clone 2
  c=274,37 ng/µl
• P318 = pSB1C3_Rep68_ins clone 1
  c=491,69 ng/µl
• P319 = pSB1C3_Rep68_ins clone 2
  c=498,71 ng/µl

Test digestion:

Incubation time: 1 h, Incubation temperature: 37° for P250, P314 and P315
Incubation time: 45 min, Incubation temperature: 37° with HindIII for P316, P317, P318 and P319 ; 45 min, Incubation temperature: 60° with BstEII for P316, P317, P318 and P319 >br /> Preparation of gel:
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 120 Volt, running time: 45 minutes

• Marker: GeneRuler ladder mix

Comment: The P314 and P315 looks like the control vector. The RepCap Vector_SDM_InsPvuII insertion didn´t work. The P316, P317, P318 and P318 was cut with the "wrong" enzymes. The same fragment size as the original vector appeared. Next time cut with EcoRI.

Midi-Prep of pSB1C3_VCK_Bla

Investigators: Chris W.

Comment: Midi-Preps of P 320, B257

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

103. labday 28.08.2010

Mini-Prep of pSB1C3_NLS

Investigator: Stefan
comments: No glycerol stock and test digestion was performed because bacteria culture inocculated from glyerol stock (B233).

Mini-Prep following the standard protocol
• P321 = pSB1C3_NLS (1)
  c= 180,45 ng/µl
• P322 = pSB1C3_NLS (2)
  c= 187,26 ng/µl

COMMENT (Patrick): These mini-preps will be thrown away because B233 was used for inoculation due to a wrong entry in our plasmid excel sheet. B233 this is NOT the sequenced clone ! We sequenced B234/P282. Therefore 2x10 ml DYT + Cm were inoculated with B234.

Cell culture

Harvest viral Stock and Transductiot of HT and A431 cells

1. P169: 100.000 cells
2. P169: 200.000 cells
3. P169: 300.000 cells
4. P169: 400.000 cells
5. P169: 500.000 cells
6. P169: 600.000 cells
7. P169: 700.000 cells
8. P262: 100.000 cells
9. P262: 200.000 cells
10. P262: 300.000 cells
11. P262: 400.000 cells
12. P262: 500.000 cells
13. P262: 600.000 cells
14. P262: 700.000 cells

200.000 cells per well

Seeding AAV293 for Transfection at 30.8

8 plates with 200.000 cells each were seeded

Picking clones from the ViralBricks

Investigator: Volker

Clones from all 14 transformations of the ViralBricks were picked. Because the probability that the ligation worked and that the synthesized oligos do not conatin mutations is quite bad I picked 8 to 10 clones from each ligation, resulting in approximately 150 clones at all. The clones were used to inoculate 5ml of a mixture of 60%LB-media nd 40%DYP-media because our stocks were not sufficient for this experiment.

104. labday 29.08.2010

P282/B234 mini-prep

Investigator: Patrick ... was performed according to the standard protocol.
Labelling:

• P323, pSB1C3_NLS clone 2.1, 129 ng/µl
• P324, pSB1C3_NLS clone 2.2, 141 ng/µl

Digestion of pSB1C3_NLS (P324)

Investigator: Patrick

The NLS was cut out with NgoMIV and EcoRI, therefore the expected size of the hoped-for fragment is 55 bp. About 30 ng DNA can be detected in a 1% gel. According to my calculation there will be maximal 80 ng insert.
55bp/2019bp = 0,027
0,027x140 ng/µl = 3,81 ng/µl
80 ng/3,81 ng/µl = 21 µl

Digestion: 21 µl pSB1C3_NLS (P324), 2,5 µl EcoRI, 2,5 µl NgoMIV, 3 µl Buffer 4, 1 µl H2O.
Digestion Time: 2 h 20 minutes.

The 2% agarose gel was loaded with 36 µl digesteion product including 6 µl 30% glycerol solution. I did not use loading dye because i to prevent that the dye overlay the mutual 55 bp insert.

The gelextraction was performed according to the the standard protocl. The resolution of the picture ist not sufficient to be able to see the band clearly as the gel was put onto the UV-light table a very thin band with the expected size could be seen.
DNA concentration measurement: 3,28 & 7,7 ng/µl (two measurements). Especially the second value is doubtable because according to my calculation above the insert conccentration cant be higher than 3,81 ng/µl. Labeled: NLS, put into 4°C room.

New digestion over night following a gelrun tomorrow: 23 µl pSB1C3_NLS (P324), 2 µl EcoRI, 2 µl NgoMIV, 3 µl Buffer 4.

Harvesting cultures from the ViralBricks

Investigator: Volker

All approximately 150 cultures were pelletted (twice 2 ml) and the supernatant thrown away. The pellets were freezed and the rest of the culture stored in the 4°C room. These pellets will be preped, test digested and sequenced subsequently until right clones are identified.

105. labday 30.08.2010

BioBrick assembly of pSB1C3_leftITR_pTERT & pSB1C3_ß-globin_YFP_hGH_rITR

Investigator: Achim

comment: final assembly of a vector containing all aav elements & the tert promoter
Digestion:

Preparation of gel:
0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time: 45 minutes

Expected sizes of constructs:

• pSB1C3_ß-globin_YFP_hGH_rITR: ~4000 bp
• pSB1C3_leftITR_pTERT: 2896 bp, ~640 bp

The corresponding bands were cut out and Gel-Extraction was performed according to protocol.

concentrations measured via NanoDrop:

• pSB1C3_ß-globin_YFP_hGH_rITR: 34.73 ng/µl
• pSB1C3_leftITR_pTERT: 9.48 ng/µl

Ligation:

T4 ligation was performed according to protocol.

Volumes used:

• vector: 2,9 µl
• insert: 5,1 µl

Transformation:

bacterial strain used: XL1bB

antibiotic used for plate: chlorampenicol

Transformation was performed according to protocol.
Plate was prepared and put in 37°C room.

Continuation: Digestion of pSB1C3_NLS (P324)

Investigator: Patrick

The gelextraction was performed with QIAEX II Gel Extraction Kit which should also allow to extract fragments smaller than 70 bp (at least 40 bp). According to the QIAGEN Gel Extraction Kit the lenght of the DNA fragment should be about 70 bp. Unfortunately the DNA fragment was blurred so a quite large piece had to be cut out.

The extraction yielded 4,26 and 3,77 ng/µl (two measurements) but this sample also quite polluted. ... put into freezer

CD biobrick production

Investigator: Kira
NA samples were diluted 1:100

PCR program:

Digestion of plasmid backbone:

c (pSB1C3) = 151, 1 ng/ µl

incubation @ 37 C for approx. 2 h

Digestion of PCR product:

incubation @ 37 C for approx. 2 h

1% agarose gel

Ligation
T4 ligase was used
1 ul T4 Buffer
1 ul T4 Ligase
9 ul (6,9 ul vector+ 1,1 ul insert) DNA-mix

incubation over-night @ 18C

Repetition of test digestion of pSB1C3_Rep40_RC_Insert and pSB1C3_Rep68_RC_Insert.

Investigator: Chris L.

• P316 = pSB1C3_Rep40_ins clone 1
  c=302,22 ng/µl
• P317 = pSB1C3_Rep40_ins clone 2
  c=274,37 ng/µl
• P318 = pSB1C3_Rep68_ins clone 1
  c=491,69 ng/µl
• P319 = pSB1C3_Rep68_ins clone 2
  c=498,71 ng/µl

Test digestion:

Incubation time: 1 h, Incubation temperature: 37° >br /> Preparation of gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time: 45 minutes

Comment: Test digestion looks good. Clones 2 of each plasmid (P317 and P319) were sent in for sequencing with VR2 Primer.

Midi-Prep of pAAV_RC_insertparts clone1

Investigators: Chris W.

Comment: Midi-Preps of B208

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

FACS analysis, Seeding HT1080 and AAV293

Investigators: Kerstin, Adrian

• FACS analysis of Transduction from 28.08.2010
• Seeding HT1080 cells for Transduction (31.08.2010): 5x 6-well plates, 270.000 cells per well
• Seeding AAV293 cells for Transfection (01.09.2010): 10 plates, 300.000 cells per well

Mini-Preps and test digestion of Loop insertion BioBricks

Investigator: Anna

Comment: Two clones of each ViralBrick were prepared and test digested.

Mini-Preps:

Test digestion:

1% Agarose gel: 1 g Agarose, 100 ml TAE, 6 µl Gelred
Marker: Gene ruler ladder DNA mix, 5 µl

Used enzymes: SalI, BamHI (in general, SDS has to be added because of the strong binding of enzymes to DNA!)

Comment: Test digestion had no significant results. In addition, one sample was forgotten, it was not clear which one (description makes no sense). Test digestion has to be repeated.

Repetition of subcloning Cap-insert into pAAV_RC

Investigator: Stefan

Comments: There are still problems with BspMI. It was assumed that longer digestion could improve the results, therefore digestion with BspMI will be performed overnight.
P.S: Welcome back Hanna!

Digestion of vector and insert:

• Insert: pMA_RepCap Vector_SDM_InsPvuII clone 1 (P211)
• Vector: pAAV_RC_ins-rep clone1 (P250)
  

A two-step digestion was performed:

1st step

2nd step

Digestion will be performed overnight and continued tomorrow.

106. labday 31.08.2010

Cloning ZEGFR:1907 and 6xHis_middlelinker into pCerulean

Investigator: Hanna

Comment: In order to fuse the His-Tag to VP2, the His-Tag-Middlelinker construct will be cloned into the pCerulean plasmid (contains CMV promoter and SV40 terminator) today. In the next step, VP2 will be cloned behind the His-Tag-Middlelinker.
In addition to that the Affibody (ZEGFR:1907) will be also cloned into pCerulean (without linker). Also here, the next step will be fusing VP2 behind the this targeting molecule.

Practical Cloning:

• plasmid:
  • Vector: name: pCerulean; number: P273
  • Insert: name: pSB1C3_6xHis_middlelinker (P310) + pSB1C3_Zegfr:1907 (because P267 was empty, P285 was used! See lab day 23.08.!)
• new vector name: pCerulean_6xHis_middlelinker + pCerulean_Zegfr:1907
• buffer used: 4; Restriction-enzymes used: XbaI and PstI

Comments: Because the P267 tube was empty (!), P285 was used which was prepared by Jessy - I don't know whether this is a trafo of the glycerol stock? Sequenced?

Digestion

• Incubation: 1.5 h

Agarose-Gel:

0.8 g Agarose, 55 ml TAE (1.45 %), 3 µL GELRED, at 115 Volt, running time: 35 minutes

• Marker: GeneRuler ladder mix

Gel extraction

Gel measurement:

Comment: The DNA concentrations were very bad, which could be due to the fact, that the 6xHis_middlelinker construct's size is just 86 bp and the affibody's size is just 210 bp...

Ligation

1. pCerulean_6xHis_middlelinker

2. pCerulean_Zegfr:1907

Trafo

Trafo was performed following the standard protocol. Cells: XL1b (60 µL), DNA amount per sample: 3 µL; cells were plated onto Kanamycin-plates.

PCR of SV 40 terminator

Investigator: Bea

Comment: In order to obtain the SV40 poly adenylation site and clone it into the iGEM standard plasmid pSB1C3 a PCR needs to be performed with the pEGFP-C1 which contains the SV40 terminator.

• Plasmid used for PCR: pEGFP-C1 (P230)
• 1:1000 dilution of P230 has been prepared
• Plasmid used as vector: pSB1C3_CFP1 (P51.2)

PCR (was performed following the standard protocol)

PCR program:

After the PCR was performed, the sample was loaded on a 1.5% agarose gel and run for 30 minutes. As it can be seen in the picture below the expected size of the PCR product (260bp) can be confirmed. The lower band corresponds to the added primers and does not seem to be an additional band.

For cloning the obtained fragment into the pSB1C3 standard vector, the construct need to be digested with the same restriction enzymes as the PCR product will be cut with. In this case the primers were designed with XbaI overhang for the prefix, and with SpeI-NotI-PstI overhang for the suffix. Therefore the vector will be cut with XbaI and PstI. The sample were incubated at 37 C for approx. 1.5 h

• Plasmid used: pSB1C3_CFP (P51.2) c=150ng/µL
• Add BSA
• XbaI and PstI were used

Digestion of vector:

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

After the PCR has been cut out of the gel and purified with the Qiagen Gel Extraction Kit obtaining a concentration of 45 ng/µl measured with the Nanodrop, the complete PCR product volume of 29 µL was digested with XbaI and PstI for 2 hours.

Digestion of the PCR product SV40

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

The digested PCR product SV40 was purifed with the PC Purification Kit and concentration was measured.

• c_SV40= 31,5 ng/µl
• c_pSB1C3= 7,74 ng/µL

For T4 ligation of the two fragments, the needed volumes of insert (SV40) and vector (pSB1C3) were calculated with the labtools program. The total volume DNA (vector/insert) mix will be 8 µL.

• v_SV40= 0,67 µL
• v_pSB1C3= 7,33 µL
• v_{T4 buffer}= 1 µL
• v_{t4 ligase}= 1 µL

The ligation reaction was carried out for 40 minutes at room temperature. The ligated plasmid was transformed into XL1-Blue cells and plated on agar plates containing the appropriate amount of chloramphenicol.

Cloning of NLS into pCerulean_VP1up

Investigator: Anissa

Comment: NLS was already 2 times cut of the pSB1C3_NLS and purified by Patrick (NLS 1: of the 29.08.10, NLS 2: of the 30.08.10) . Now, pCerulean_VP1up will be digested and NLS will be ligated into it.

• Digestion of pCerulean_VP1up (p303)
  
  

  components	Vector
  DNA	2,6
  BSA (10x)	1,5
  Buffer 4 (10x)	1,5
  Enzyme PstI	1
  Enzyme AgeI	1
  H2O	7,4
  Total volume	15

  
  

• Gel:
  A 1% gel was made and the sample was running 30 minutes.
  
  Afterwards a gelextraction according the qiagen-protocol was performed.
  
  
• Ligation:
  

  components	Volume /µl	concentration /ng/µl
  NLS 2 insert of 30.08.10	1,51	3
  NLS 1 insert of 29.08.10	1,51	3
  pCerulean_VP1up	6,49	29

  
  


• Transfomation: was performed into XL1 blue on a kanamycin plate according the standard protocol.