Team:Freiburg Bioware/NoteBook/Labjournal/September2

PCR program:

Digestion of the vector:

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

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

Gelextraction:

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

• pSB1C3 (P320): c= 8,5 ng/µl

Digestion of the PCR-products:

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

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

• PCR of P432: c= 6,4 ng/µl

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

The Ligation was performed using the following amounts of DNA:

• Vector Volume: 0,92 µl
• Insert Volume: 7,08 µl

Transformation:

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

Picking clones of pSB1C3_CFP_email

Investigator: Anna

Three clones were picked (10 ml DYT, 10 µl Chloramphenicol).

MiniPreps and Test digestion of pSB1C3_CMV_VP123_capins

Investigator: Anna

Test digestion:

Expected size (CFP): 681 bp

Comment: Clone 1 was sent for sequencing with the following primers: VF2, 4000 for, 4200 rev, 2800 for and 2800 rev.

MiniPreps of 587 Z34C ViralBricks and pSB1C3_p40

Investigator: Anna

Comment: Both clones were sent for sequencing.

Re-Trafo of DARPin

Investigator Stefan

Comment: A Re-Trafo of the synthesized DARPin was performed using XL1b cells.

130. labday 25.09.2010

Sequencing of VB: 13.1, 13.4 (approach w. dephosphorylated vector, second clone picking round

Investigator: Achim

The two clones that were sequenced both contained a deletion in the same position in the insert. We really seem to have bad luck picking clones. Hopefully our PCR attempt works better.

Miniprep of serveral constructs

Investigator: Jessica

Glycerol stocks were prepared:

• B489 = pBAD_EGFP_His clone1
• B490 = pBAD_EGFP_His clone2
• B491 = pBAD_EGFP_His clone3
• B492 = pSB1C3_CFP_email clone 1
• B493 = pSB1C3_CFP_email clone 2
• B494 = pSB1C3_CFP_email clone 3

Mini-Prep was performed according to the standard protocol

• P588= pBAD_EGFP_His clone1 c=71,0 ng/µl
• P589 = pBAD_EGFP_His clone2 c=74,8 ng/µl
• P590 = pBAD_EGFP_His clone3 c=58,3 ng/µl
• P591 = pSB1C3_CFP_email clone 1 c=132,7 ng/µl
• P592 = pSB1C3_CFP_email clone 2 c=168,9 ng/µl
• P593 = pSB1C3_CFP_email clone 3 c=153,6 ng/µl

Test digestion:

Expected size : bp

Comment: test digestion of P591-593 and P583-585 (pSB1C3_P40) is just an attempt because I can't find any sequences in geneious.P583 looks well. P591, P588 and P583 will be sent for sequencing on monday.

Sequencing results of pSb1C3_Affibody_middlelinker_EGFP_His

Comment: there was anywhere a problem in the cloning process. approach will be repeated today. there is pBAD_EGFP_His, affibody_middlelinker will be cloned in, also in pSB1C3_EGFP_His

Sequencing result of pSB1C3_CMV_VP123_capins_clone1

Investigators: Volker, Anna

For test digestion go to labday 24.09.10

Comment: The following primers were used for sequencing: VF2, 4000 for, 4200 rev, 2800 for and 2800 rev. There is a point mutation in the non-coding sequence, which should not influence the performence of the construct, so it can be used for further cloning steps.

Cloning of 453 and 587 ViralBricks into pSB1C3_CMV_VP123_capins

Investigators: Achim, Anna

• Vector name: pSB1C3_CMV_VP123_capins P580
• Inserts: different ViralBricks (see pdf underneath), pSB1C3_VCK_Bla P320
• buffer used: 4

Update: ViralBricks ready to use:

media:Freiburg10_Production of ViralBricks_new.pdf

Digestion of vector and inserts:

media:Freiburg10_Cloning of ViralBricks into pSB1C3_CMV_VP123_capins.pdf

The vector p580 was dephosphorylated.
Two gels were prepared with 1 g Agarose, 100 ml TAE (1%) and 6 µl GELRED; at 120 Volt, running time:45

Gel run:
Loading dye(6X), 6 µl
Marker used: GeneRuler ladder mix (Fermentas), 3 µl

Expected sizes of fragments: see Pdf under Gel extraction and overnight T4 Ligation

Gel extraction and overnight T4 Ligation:

media:Freiburg10_Cloning of ViralBricks into pSB1C3_CMV_VP123_capinsII.pdf

Sequencing results: pSB1C3_001_VP2/3_HSPG-KO

Investigator: Hanna

Comment: In order to knock down the natural tropism of the virus, the HSPG knock-out motif (two aa exchanges) was cloned into VP2/3. This fragment can then be used for the VP2 N-terminal fusion or VP1 insertion approaches.


Conclusion: Sequencing results looked well: The HSPG-KO ViralBrick was cloned successfully into VP2/3.

Cloning of pBAD_Affibody_middlelinker_EGFP_His and pSB1C3_Affibody_middlelinker_EGFP_His

Investigator: Jessica
Comment: There was a mistake in cloning pSB1C3_Affibody_middlelinker_EGFP_His so there is just pSB1C3_EGFP_His. in this plasmid will be cloned Affibody_middlelinker, also in pBAD_EGFP_His

• Vector: name: pBAD_EGFP_His P588
• Vector: name: pSB1C3_EGFP_His P565
• Insert: name: pSB1C3_ZEGFR:1907_middlelinker_ P290
• new vector name: pBAD_ZEGFR:1907_middlelinker_EGFP_His P
• new vector name: pSb1C3_ZEGFR:1907_middlelinker_EGFP_His P
• buffer used: 4
• DNA concentration (vector): 71,0 ng/µl / 252,7 ng/µl; DNA concentration (insert): 227,4 µg/µl

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

Gel extraction:

T4 Ligation pBAD:
Volume vector: 3,5 µl
Volume insert: 4,5 µl


T4 Ligation pSB1C3:
Volume vector: 2,3 µl
Volume insert: 5,7 µl

131. labday 26.09.2010

Continuation of cloning of pBAD_Affibody_middlelinker_EGFP_His and pSB1C3_Affibody_middlelinker_EGFP_His

Transformation:

BL21 cells were used - in 37°C at 8.30am

• pBAD_Affibody_middlelinker_EGFP_His
• pSB1C3_Affibody_middlelinker_EGFP_His
  

Comment:pSB1C3_Affibody_middlelinker_EGFP_His was not successful (no colonies on plate), will be repeated on monday

Picking clones of different constructs

Investigator: Anna

Clones were picked of:

• pBad_Affibody_middlelinker_EGFP_His
  
• pSB1C3_001_RC_InsRepCap_KpnIback_RFC10
  
• Darpin (ReTrafo)
  
• pSB1C3_453_BAP (p402, ReTrafo)
• pSB1C3_587_KO_RGD (p390, ReTrafo)
• pSB1C3_587_KO_His (p391, ReTrafo)

In addition, preparations for Mini-Preps of several ViralBricks were done:

• B289: 587_BAP
• B270: 587_KO_BAP
• B271: 587_RGD
• B272: 453_His
• B273: 587_His
• B274: 587_KO_empty
• B488: 453_Z34C

Comment: Inoculating of 10 mL DYT medium containing 10µL chloramphenicol (except pBad with Amp).
There were no clones on the plate with pSB1C3_Affibody_middlelinker_EGFP_His.

Continuation of cloning of 453 and 587 inserts into pSB1C3_CMV_VP123_capins

Investigator: Achim, Anna

Transformation:

Trafo was performed according to the standard protocol using XL1b cells. The cells were plated on agar plates containing chlorampenicol and stored in the 37°C room.

Colony PCR of remaining 587 constructs

Investigator: Achim, Anna

Comment: New clones of the remaining 587 constructs were picked (from new cloning approach with T4 Ligation and dephosphorylated vector, see labdays 15.09. and 22.09.).

Constructs:

• 12: 587_Z34C
• 13: 587_KO_Z34C
• 14: 587_Z34C_KO_Spacer

Clone 13.3, 14.1 and 14.3 will be sent for sequencing.

Sequencing results: pCerulean_Zegfr:1907_VP2/3

Investigator: Hanna

Sequencing results revealed that VP2/3 (unmodified!) was successfully fused to the Affibody (without linker).
Next step: Fusion of VP2/3-HSPG-KO to the C-terminus of the Affibody.

N-terminal VP2-Fusion & VP1 Insertion: Fusion of VP2/3_HSPG-KO

Investigator: Hanna

Comment: In order to target tumor cells with our AAV2 two VP2 fusion strategies were figuered out: The so called VP2-fusion and VP1 insertions. These approaches were successfully cloned and are now tested in cell culture. In order to prevent targeting of other (healthy) cells, the natural tropism of the virus needs to be knocked down. For this purpose a ViralBrick, "587-KO_empty", was designed and cloned into pSB1C3_001_VP2/3_Capins.
Today this VP2/3_HSPG-KO sequence will be fused to the N-terminus of the Affibody, CFP/mVenus and His-Tag of both, the N-terminal fusion to VP2 and the VP1 insertion, approach.

DIGESTION:

1. pCerulean_CFP_MiddleLinker (P407)
2. pCerulean_Z_EGFR:1907_MiddleLinker (P408)
3. pCerulean_Z_EGFR:1907_SEG (P409)
4. pCerulean_Z_EGFR:1907_ShortLinker (P410)
5. pCerulean_Z_EGFR:1907_LongLinker (P412)
6. pCerulean_6xHis_MiddleLinker (P374)
7. pCerulean_VP1up_NLS_Z_EGFR:1907 (P422)
8. pCerulean_VP1up_NLS_6xHis (P424)
9. pCerulean_VP1up_NLS_mVenus (P426)
10.1 pSB1C3_001_Vp2/3_HSPG-KO (P579)
10.2 pSB1C3_001_Vp2/3_HSPG-KO (P579)

Incubation: 2 hours, 37°C.
Agarose gel: 0.8%

• Marker: GeneRuler ladder mix

LIGATION:

TRAFO:
Trafo was performed following our standard protocol. Cells: XL1b, DNA: 2µL.
Plates will be stored over night @ the 37°C room.

Next step: Picking 2 clones of each construct, colony PCR!

132. labday 27.09.2010

Sent for sequencing

Investigator: Jessica

• P591 pSB1C3_email: VR2
• P583 pSB1C3_P40: VR2
• P588 pBAD_EGFP_His: pBAD-FP
• P596 pBAD_Affibody_middlelinker_EGFP_His: pBAD-FP / pTrcHis-RP

Picking clones of VP1 insertion and VP2 fusion HSPG-KO approaches

Investigator: Hanna
2 clones were picked of each ligation approach = 18 falcons (Kanamycin).

To do: Mini-Prep and test digestion - sequencing.

MiniPreps of different constructs

Investigator: Anna

The following constructs were prepped according to the standard protocol:

• pSB1C3_453_BAP (p402, ReTrafo)
• pSB1C3_587_KO_RGD (p390, ReTrafo)
• pSB1C3_587_KO_His (p391, ReTrafo)
• B289: 587_BAP
• B290: 453_RGD
• B270: 587_KO_BAP
• B271: 587_RGD
• B272: 453_His
• B273: 587_His
• B274: 587_KO_empty
• B488: 453_Z34C

• B473:

Miniprep of serveral constructs

Investigator: Stefan

Glycerol stocks were prepared:

• B489 = pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 1
• B490 = pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 2
• B491 = pBAD_Affibody_middlelinker_EGFP_His clone 1
• B492 = pBAD_Affibody_middlelinker_EGFP_His clone 2
• B493 = pBAD_Affibody_middlelinker_EGFP_His clone 3

Mini-Prep was performed according to the standard protocol

• P594= pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 1 c= 140,9 ng/µl
• P595 = pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 2 c= 117,5 ng/µl
• P596 = pBAD_Affibody_middlelinker_EGFP_His clone 1 c= 101,4 ng/µl
• P597 = pBAD_Affibody_middlelinker_EGFP_His clone 2 c= 92,9 ng/µl
• P598 = pBAD_Affibody_middlelinker_EGFP_His clone 3 c= 92,1 ng/µl

Test digestion:

Comment:

Re-Trafo of pSB1C3_pTAV (P5)

Investigator: Stefan

Comment:After inocculating from glycerol-stock, the plasmid showed strange results during digestion. Therefore a re-trafo will have to be performed to replace the stock.

Repetition: Cloning of P5_TATAless downstream of pSB1C3_001_RC_insrepcap_KpnI_back and P5 upstream of pSB1C3_001_RC_insrepcap_KpnI_back

Investigator: Stefan
Digestion of the constructs:

• P594 = pSB1C3_001_RC_insrepcap_KpnI_back c=344,5 ng/µl
• P594 = pSB1C3_001_RC_insrepcap_KpnI_back c=344,5 ng/µl
• P586 = pSB1C3_pTAV2 (P5)clone 1 c=137,37 ng/µl
• P587 = pSB1C3_pAAV_RC (P5TATAless)clone 3 c=191,61 ng/µl

Results:

Comment:P5 and P5TATAless do not fit the expected results. For P5TATAless, a new digestion using the P184 plasmid was performed. For P5 no old plasmid stock was available, therefore a re-trafo will have to be performed.

Comment:No gel picture available because computer crashed completely...

Gel-Extraction:
Gel-Ex was performed according to protocol. The following concentrations were measured:

• 594down c= 29,9 ng/µl
• 184down c= 3,8 ng/µl

Ligation:
The following DNA mixes were used for ligation:

• 432down V= 4,48 µl
• 184down V= 3,52 µl

Transformation:
The transformation was performed according to standard protocol using BL21 cells. They were plated on agar plates containing chloramphenicol and stored in 37°C room overnight.

Picking clones of VP1 insertion and VP2 fusion HSPG-KO approaches

Investigator: Hanna
2 clones were picked of each ligation approach = 18 falcons (Kanamycin).

To do: Mini-Prep and test digestion - sequencing.

Picking clones of VP1 insertion and VP2 fusion HSPG-KO approaches

Investigator: Hanna
2 clones were picked of each ligation approach = 18 falcons (Kanamycin).

To do: Mini-Prep and test digestion - sequencing.

133. labday 28.09.2010

Cloning of the ViralBricks BAP-HSPG-ko and His-HSPG-ko into pSB1C3_VP2/3

Investigator: Bea

Comment: After discussing the finals constructs, we decided to clone HSPG-KO with BAP and His motifs into the N-terminal approaches which means that the plasmids pCerulean_Targeting_VP2/3 contains His and BAP ViralBricks integrated in the 587 loop, respectively. Therefore I started the first cloning step in order to obtain the fully assembled targeting construct which can be cotransfected with the plasmids containing rep and cap genes (with or wihtout HSPG-ko)

Digestion of the constructs:

• P604 = pSB1C3_001_587-ko_BAP
• P606 = pSB1C3_001_587-ko_His
• P525 = pSB1C3_001_VP2/3capins

Loading plan of a 1% agarose gel:

M    P525    

Loading plan of a 2% agarose gel:

M    P604    P606

Expected sizes of the digested products are:

• P525= 48,3800 bp
• P604= 2216, 81bp
• P606 = 2216, 108bp

Results:

After gel extraction has been performed, the 2µL of each ligated plasmids were transformed into BL-21 cells.

Test digestion: ViralBricks in pSB1C3_CMV_VP123_capins

Investigator: Achim

• We digested preps of all our 453 Inserts with BamHI & SspI and all our 587 Inserts with SalI and PvuII. That way we get bands of ~50 bp difference between our loop inserts and the wt loop region. (Eg. 453 BAP: 455 with insert, 396 without insert.)

• Nomenclature as in previous VB assays.

• Clone 11.2 is missing , I forgot to inoculate it.

• P580 (pSB1C3_CMV_VP123_capins)

The framed samples were midiprepped and will be used for transfection

Sequencing result of pSB1C3_email and pBAD_Affibody_middlelinker_EGFP_His

Investigator: Jessica
1. pSB1C3_email:

2. pBAD_Affibody_middlelinker_EGFP_His:

Preparation of PBS Buffer and PBS Buffer with Imidazole

Investigator: Jessica

The following protocol was used:

• dissolve the following in 800ml ddH₂O:
  • 8g of NaCl
  • 0.2g of KCl
  • 1.44g of Na₂HP0₄ (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

• dissolve the following in 180ml ddH₂O:
  • 6,4g of NaCl
  • 0,16g of KCl
  • 1,15g of Na₂HP0₄ (1.69 because of the crystal water in the reagent)
  • 0,19 g of KH₂PO₄
  • 3,4g of Imidazole
• adjust to pH 7.4
• adjust to 200ml
• steril filtrate the solution

ViralBrick Sequencing results: pSB1C3_587_KO_Z34C and pSB1C3_587_KO_Z34C_Spacer

Investigator: Achim

Sequencing showed that the PCR reaction we designed to remove the mutations in the z34c insert were successful! Both pSB1C3_587_KO_Z34C and pSB1C3_587_KO_Z34C_Spacer are correct. We hadn't sent the 587_Z34C for sequencing because the colony PCR hadn't shown any bands. But because this construct is less important for cell culuture, we won't continue working on this construct.

Cloning of Darpin into pCerulean_Vp1up_NLS and pSB1C3

Investigator: Achim

• The Darpin needs to be cloned into pSB1C3 for submission to the parts registry as well as into pCerulean for expression.

• Darpin ( cut with NgOMIV & SpeI
• Vectors cut with AgeI & SpeI

• • -> Results in fusion protein

Cloning of pSB1C3_001_CMV and pSB1C3_001_Vp2/3_HSPG-Ko_CMV_ZEGFR:1907

Investigator: Anissa
Comment: By working with three-fragment-ligation, CMV and ZEGFR:1907 will each be cut out of pSB1C3, will be ligated together and into the vector pSB1C3_001_Vp2/3_HSPG-Ko. Additionally, CMV will be cloned freom pSB1C3 into pSB1C3_001

• Vector: name:pSB1C3_001_Vp2/3_HSPG-Ko P613 c=273,6 ng/µl
• Vector: name: pSB1C3_001_bla P320 c=408 ng/µl
• Insert: name: pSB1C3_CMV P146 c=155,6 ng/µl
• Insert: name: pSB1C3_ZEGFR:1907 P268 c=162,5 ng/µl
• new vector name: pSB1C3_001_CMV
• new vector name: pSB1C3_001_Vp2/3_HSPG-Ko_CMV_ZEGFR:1907
• buffer used: 4

0,5 g Agarose, 55 ml TAE (1%), 1,5 µl GELRED , at 115Volt

Gel extraction:

T4 Ligation pSB1C3_001_CMV:
Volume vector p320: 4,63 µl
Volume insert p146: 3,37 µl


T4 Ligation pSB1C3_001_Vp2/3_HSPG-Ko_CMV_ZEGFR:1907:
Volume vector p613: 2,9 µl
Volume insert p268: 3 µl
Volume insert p146: 3 µl

Transformation:
Was performed in BL21 according the standard protocol

CD assembly with new suffix primer

Investigator: Kira

• PCR program:

no PCR bands are visible on the gel..--> PCR reaction failed.

test digestion of all CD constructs

Investigator: Kira

Comment: Because the PCR failed also with new suffix primer, we decided to check if the DNA samples are ok. 3 different CD samples were used as DNA samples.
1_original CD with iGEM binding sites p(223)
2_CD with SDM of PstI
3_CD with SDM of PstI and NgoMIV (p290)

Cloning of pSB1C3_Affibody_middlelinker_EGFP_His

Investigator: Jessica
Comment: There was a mistake in cloning pSB1C3_Affibody_middlelinker_EGFP_His so there is just pSB1C3_EGFP_His. in this plasmid will be cloned Affibody_middlelinker_EGFP_His

• Vector: name: pSB1C3_EGFP_His P565
• Insert: name: pBAD_Affibody_middlelinker_EGFP_His P596
• new vector name: pSb1C3_ZEGFR:1907_middlelinker_EGFP_His P
• buffer used: 4
• DNA concentration (vector): 252,7 ng/µl; DNA concentration (insert): 101,4 µg/µl

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

Gel extraction:

T4 Ligation:
Volume vector: 2,9 µl
Volume insert: 5,1 µl

Transformation:

BL21 cells were used

Sequencing analysis of pSB1C3_001_RC_IRCK, pSB1C3_001_RC_IRCK_VP1-ko_P5tataless and pSB1C3_001_RC_IRCK_VP2-ko_P5tataless

Investigator: Stefan

Because all these constructs were cloned via PCR out of the pAAV vector, a complete sequencing was performed:

P548 pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 1:
Sequence could be verified, can be used for further cloning steps!

P568 pSB1C3_001_RC_InsRepCap_KpnIback_VP2-KO_P5tataless clone 2:
Sequence could be verified, can be used for further cloning steps!

P594 pSB1C3_001_RC_InsRepCap_KpnIback_RFC10 clone 1:
Sequence could be verified, can be used for further cloning steps!

Cloning of HSPG_KO_empty into pSB1C3_001_RC_IRCK_VP1-ko_P5tataless and pSB1C3_001_RC_IRCK_VP2-ko_P5tataless

Investigator: Stefan

Vector name:

• pSB1C3_001_RC_IRCK_VP1-ko_P5tataless (P548)
• pSB1C3_001_RC_IRCK_VP1-ko_P5tataless (P568)

Insert name:

• VB:pSB1C3_001_KO_empty(P611)

Gel:
for vector:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt

for insert:
1 g Agarose, 50 ml TAE (2%), 3 µl GELRED , at 105 Volt

Gel extraction:
Was performed according to protocol.

T4 Ligation:

VP1-ko:

• Volume VP1-ko: 5,57 µl
• Volume insert: 2,43 µl

VP2-ko

• Volume VP2-ko: 5,04 µl
• Volume insert: 2,96 µl

Transformation:
Was performed according to standard protocol using BL21 cells.

Cloning of 587_KO_Z34C and 587_KO_Z34C_Spacer into pSB1C3_001_CMV_VP123_capins

Investigator: Anna

Comment: Todays sequencing results of 587_KO_Z34C and 587_KO_Z34C_Spacer looked well, so they will be cloned into pSB1C3_CMV_VP123_capins. Finally produced ViralBricks:

media:Freiburg10_Production of ViralBricks_End version.pdf

• Vector name: pSB1C3_CMV_VP123_capins P580
• Insert name:

1. pSB1C3_587_KO_Z34C P599
2. pSB1C3_587_KO_Z34C_Spacer P600

• buffer used: 4

• DNA concentration (P580): 377,29 ng/µl;
• DNA concentration (P599): 102,5 µg/µl
• DNA concentration (P600): 90,4 µg/µl

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

Gel extraction:
Was performed following the standard protocol.
DNA-concentrations [ng/µl]:

c(vector)= 18,88
c(587_KO_Z34C)= 9,74
c(587_KO_Z34C_Spacer)= 1,72

T4 Ligation:

587_KO_Z34C:
Volume vector: 1,5 µl
Volume insert: 6,5 µl

587_KO_Z34C_Spacer:
Volume vector: 4,6 µl
Volume insert: 3,4 µl

Trafo:
Was performed following the standard protocol using BL21 cells.

Mini-Prep and Test-digestion of VP1 insertion and VP2 fusion HSPG-KO approaches

Investigator: Hanna

Comments: Yesterday, 2 clones of each construct were picked. Today mini-prep and test digestion will be performed of one of each approach - the other clones will be spinned down and stored at -20°C - waiting for test digestion and sequencing results.

Mini-Prep was performed, following the standard protocol.

New plasmid names:
P617: pCerulean_CFP_MiddleLinker_VP2/3_HSPG-KO
P618: pCerulean_ZEGFR:1907_MiddleLinker_VP2/3_HSPG-KO
P619: pCerulean_ZEGFR:1907_SEG_VP2/3_HSPG-KO
P620: pCerulean_ZEGFR:1907_ShortLinker_VP2/3_HSPG-KO
P621: pCerulean_ZEGFR:1907_LongLinker_VP2/3_HSPG-KO
P622: pCerulean_6xHis_MiddleLinker_VP2/3_HSPG-KO
P623: pCerulean_VP1up_NLS_ZEGFR:1907_VP2/3_HSPG-KO
P624: pCerulean_VP1up_NLS_6xHis_VP2/3_HSPG-KO
P625: pCerulean_VP1up_NLS_mVenus_VP2/3_HSPG-KO

TEST DIGESTION:
Mastermix:

• BSA (10x): 10 µL
• Buffer 4: 10 µL
• EcoRI: 5 µL
• PstI: 5 µL
• H₂O: 37 µL

DNA: 3.3 µL + 6.7 µL Master Mix.

Conclusion: Test digestion looked well - except of P618: There is an additional band visible, which can't be correlated to any construct.
All samples were sent for sequencing.

134. labday 29.09.2010

CD assembly with new suffix primer

Investigator: Kira

• PCR program:

as DNA samples p291 and p292 with and wo DMSO were used.

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 with T4 ligase:
c(insert)= 31,35 ng/ul
c(vector)= 10,45 ng/ul
8ul DNA mix = insert 3 ul + vector 5 ul
T4 ligase 1 ul
T4 buffer 1 ul

Transformation: Anna

Miniprep of serveral constructs

Investigator: Stefan

Glycerol stocks were prepared:

• B516 = pSB1C3_001_RC_ICRK_P5tataless_RFC10 clone 1
• B517 = pSB1C3_pTAV (P5)
• B518 = pSB1C3_001_RC_ICRK_P5tataless_RFC10 clone 2

Mini-Prep was performed according to the standard protocol

• P628 = pSB1C3_001_RC_ICRK_P5tataless_RFC10 clone 1
• P629 = pSB1C3_pTAV (P5)
• P630 = pSB1C3_001_RC_ICRK_P5tataless_RFC10 clone 2

Test digestion:

Comment:First test digestion using XmaI and SpeI did not work out, thererfore, two different approaches using MngBI/PstI and SnaBI and PstI were performed. This verified successful cloning of P5tataless into the target vector.

Sent for sequencing:

pSB1C3_001_RC_ICRK_P5tataless_RFC10 clone 1 (P628) was sent for sequencing using VR2 primer.

mGMK_TK30 approaches

Investigator: Bea

Comment: Since we are still waiting for the TK30 gene ordered at Mr.Gene we decided to take another road to produce a BioBrick compatible thymidine kinase gene. Therefore, we are cloning the mGMK_TK30 fusion construct into the pSB1C3 and after removing the PstI site (see next topic) it is ready for submitting it to the parts registry. Additionally, we are fusing the mGMK_TK30 to the composite part pSB1C3-leftITR_PROMOTER_betaglobin for testing it in cell culture with pur final super construct.

Digestion of the constructs:

• P51.2 = pSB1C3_CFP
• P81 = pAAV_RFC25_mGMK_TK30
• P434 = pSB1C3_leftITR_CMV_betaglobin
• P437 = pSB1C3_leftITR_phTERT_betaglobin

Overview cloning plan:

Loading plan of a 0,8% agarose gel:

M    P51.2    P81 (digested with E+S)    P81 (digested with X+S)    P434    P437

Expected sizes of the digested products are:

• pAAV_RFC25_mGMK_TK30= 4626, 1717bp
• pAAV_RFC25_mGMK_TK30= 4626, 1717bp
• pSB1C3_CFP = 2052, 756bp
• pSB1C3_leftITR_CMV_betaglobin= 5094b
• pSB1C3_leftITR_phTERT_betaglobin= 5094b

Results:

After Gel extraction have been performed the ligation was conducetd:

Design of mutagenesis primers for deletion of PstI in mGMK_TK30

Investigator: Bea

Comment: Since we are still waiting for the TK30 gene ordered at Mr.Gene we decided to take another road to produce a BioBrick compatible thymidine kinase gene. Therefore, new primers were designed in order to remove the PstI restriction sites within the TK30 gene. The primers will be ordered tomorrow.

Cloning of Darpin into pCerulean_Vp1up_NLS and pSB1C3 Part Two

Investigator: Achim

• I accidentally plated yesterdays trafo with the wrong antibiotics and I had to digest pSB1C3 again anyway, so I prepared a new digestion of both vectors with Age & Spe.

• After watching the gel picture I realized I had digested pSB1C3 with the wrong enzymes. I should have used NgOMIV and Spe in order to cut out BLA.

• pCerulean_Vp1up_NLS was ligated with yesterdays Darpin fragment and plated on Ampicillin, the pSB1C3_BLA construct will be cloned correctly tomorrow

Cloning of pGGTBT7_Affibody_middlelinker_EGFP_His

Investigator: Jessica
Comment: pBAD is not really working, so we test another expression-plasmid

• Vector: name: pGGTBT7_MVenus P627
• Insert: name: pBAD_Affibody_middlelinker_EGFP_His 596prepared yesterday
• new vector name: pGGTBT7_ZEGFR:1907_middlelinker_EGFP_His P
• buffer used: 4
• DNA concentration (vector): 144,1 ng/µl; DNA concentration (insert): 101,4 µg/µl

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

Gel extraction:

T4 Ligation:
Volume vector: 3,2 µl
Volume insert: 4,8 µl

Transformation:

BL21 DE3 R2L(Kana+Cm), BL21 DE3(Kana), BL21DE3 R2(Kana+Cm) cells were used

Cloning of CMV promotor and Affibody into pSB1C3_001_VP2/3_capins and pSB1C3_001_VP2/3_HSPG_KO

Investigator: Anna

Comment: Cloning of CMV promotor and Affibody into pSB1C3_001_VP2/3_HSPG_KO was repeated, because it was not clear if the first attempt worked.

• Vector name:

1. pSB1C3_001_VP2/3_capins P514: 318,9 ng/µl
2. pSB1C3_001_VP2/3_HSPG_KO P613: 273,6 ng/µl

• Insert name:

1. pSB1C3_CMV P146: 155,6 ng/µl
2. pSB1C3_ZEGFR:1907 P248: 146,5 ng/µl

• buffer used: 4

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

Gel extraction:
Was performed following the standard protocol.
DNA-concentrations [ng/µl]:

c (pSB1C3_001_VP2/3_capins) = 40.9
c (pSB1C3_001_VP2/3_HSPG_KO)= 51.94
c (CMV)= 12.67
c (Affibody)= 14.44

T4 Ligation:

• Vector 1:
  

Volume vector: 2,8 µl
Volume Affibody: 2,0 µl
Volume CMV: 3,2 µl

• Vector 2:
  

Volume vector: 2,5 µl
Volume Affibody: 2,0 µl
Volume CMV: 3,5 µl

Trafo:
Was performed following the standard protocol using BL21 cells.

Cloning of CFP into pSB1C3_lITR_phTERT_beta-globin

Investigator: Anissa

• Vector name:

pSB1C3_lITR_phTERT_beta-globin P514: 333,46 ng/µl

• Insert name:

pSB1C3_CFP P146: 151,1 ng/µl

• buffer used: 4

0,5 g Agarose, 50 ml TAE (1%), 1,5 µl GELRED , at 115 Volt

Gel extraction:
Was performed following the standard protocol.
DNA-concentrations [ng/µl]:

c (pSB1C3_CFP) = 11,6
c (pSB1C3_lITR_phTERT_beta-globin)= 24,8

T4 Ligation:

• Volume vector: 3,2 µl
  
• Volume insert: 4,8 µl
  

Trafo:
Was performed following the standard protocol using BL21 cells.

Sequencing results: VP1 insertion and VP2 fusion: Fusion of VP2/3_HSPG-KO

Investigator: Hanna

Sample gallery

pCerulean_CFP_MiddleLinker_ VP2/3_HSPG-KO (P617)	pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_HSPG-KO (P618)
pCerulean_ZEGFR:1907_SEG_ VP2/3_HSPG-KO (P619)	pCerulean_ZEGFR:1907_ShortLinker_ VP2/3_HSPG-KO (P620)
pCerulean_ZEGFR:1907_LongLinker_ VP2/3_HSPG-KO (P621)	pCerulean_6xHis</sub>_MiddleLinker_ VP2/3_HSPG-KO (P622)
pCerulean__VP1up_NLS_ZEGFR:1907_ VP2/3_HSPG-KO (P623)	pCerulean_VP1up_NLS_6xHis_ VP2/3_HSPG-KO (P624)
pCerulean_VP1up_NLS_mVenus_ VP2/3_HSPG-KO (P625)

Conclusion: Except of pCerulean_Z_EGFR:1907_MiddleLinker_ VP2/3_HSPG-KO (P618), all sequencing results looked well. This means: The VP2/3-HSPG-KO motif was cloned successfully to the VP2-Fusion and VP1-Insertion approaches and are ready to be tested in cell culture via qPCR and ELISA. --> Co-transfection with pSB1C3_001_RC_iRCK_VP1or2-KO_HSPG-KO + pHelper + pSB1C3_GOI.

In addition to that, clone 2 of pCerulean_Z_EGFR:1907_MiddleLinker_ VP2/3_HSPG-KO will be preped and test digested tomorrow.

Cloning of HSPG_KO_empty into pSB1C3_001_RC_IRCK_P5tataless

Investigator: Stefan

Vector name:

• pSB1C3_001_RC_IRCK_P5tataless cl1 (P628)
• pSB1C3_001_RC_IRCK_P5tataless cl2 (P630)

Insert name:

• VB:pSB1C3_001_KO_empty(P611)

Gel:
for vector:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 110 Volt

for insert:
1 g Agarose, 50 ml TAE (2%), 3 µl GELRED , at 110 Volt

Gel extraction:
Was performed according to protocol.

T4 Ligation:

• Volume vector P628: 7,11 µl
• Volume insert: 0,89 µl

• Volume vector P630: 7,33 µl
• Volume insert: 0,67 µl

Transformation:
Was performed according to standard protocol using BL21 cells.

Mass Midi-Prep II

Investigator: Chris W.

Midi-Prep of:

453_BAP =P631
587_BAP =P632
587_KO_BAP =P633
453_RGD =P634
587_RGD =P635
587_KO_RGD =P636
453_HIS =P637
587_HIS =P638
587_KO_HIS =P639
587_KO_empty =P640
453_Z34C =P641
pSB1C3_001_RC_IRCK_VP1-ko_P5tataless =P642 =B463
pSB1C3_001_RC_IRCK_VP2-ko_P5tataless =P643 =B470

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

135. labday 30.09.2010

Cloning of Darpin into pCerulean_Vp1up_NLS and pSB1C3 Part Three

Investigator: Achim

• Clones of the pCerulean_Vp1up_NLS_Darpin construct were picked and will be prepped tonight.

• pSB1C3_001_BLA (P320) and pMA_Darpin (P614) were digested with NgOMIV and SpeI. Bands were cut out accordingly, ligated with T4 ligase and plated on Chloramphenicol.

Test digestion of pCerulean_Z_EGFR:1907_MiddleLinker_VP2/3_HSPG-KO clone 2

Investigator: Hanna

Comment: Yesterday's sequencing results revealed, that VP2/3_HSPG-KO was successfully cloned to the VP2 fusion and VP1 insertion approaches, except of pCerulean_Z_EGFR:1907_MiddleLinker_VP2/3_HSPG-KO clone 2. The chromatogram showed that fusion worked, but there was a kind of contamination in the sample - already visible in the test digestion.

• DNA = 2 µL
• BSA (10x) = 1 µL
• Buffer 4 = 1 µl
• EcoRI = 0.5 µL
• PstI = 0.5 µL
• H₂0 = 5 µL

Incubation time = 45 minutes.
0.9 % Agarose gel.

Test digestion looked well. Sample was sent for sequencing.

Cloning VP2/3_Capins and VP2/3_HSPG-KO downstream of Middle Linker

Investigator: Hanna

Comment: Besides the Affibody we also want to test and characterize the DARPin for its binding properties referring to the VP2 fusion and VP1 insertion. Therefore some preliminary cloning steps have to be performed. Today the VP2/3_insCap and VP2/3_HSPG-KO sequence will be cloned downstream of the Middle Linker for the VP2 fusion.

Practical Cloning:

• • Vector: pGA14_MiddleLinker (P301)
  • Insert: pSB1C3_VP2/3_insCap (P525) and pSB1C3_001_VP2/3_HSPG-KO (P613)
• new vector name: pGA14_MiddleLinker_VP2/3_insCap and pGA14_MiddleLinker_VP2/3_HSPG-KO
• buffer used: 4 ; Restriction-enzymes used: PstI, AgeI/NgoMIV

Digestion

• Comment: After 30 minutes of digestion I remembered that it would be adventageous to digest the plasmid backbone of the VP2/3 constructs. Therefore 0.5 µL of McsI was added to each sample - fortunately McsI does not have any restriction sites in the pGA14_MiddleLinker to which it was also added by mistake.
  

• Incubation: 20 minutes + 2 hours.

Agarose-Gel:

0.5 g Agarose, 50 mL TAE (1 %), 3 µL GELRED, at 10 Volt, running time: 45 minutes

• Marker: GeneRuler ladder mix

Gel extraction

Gel measurement:

Ligation

Miniprep of serveral constructs

Investigator: Jessica

Glycerol stocks were prepared:

• B527 pSB1C3_001_CMV_VP2/3_587-KO_Z34C_spacer clone3
• B528 pSB1C3_001_CMV_VP123_587-KO_Z34C clone1
• B529 pSB1C3_001_CMV_VP123_587-KO_Z34C clone2
• B530 pSB1C3_001_CMV_VP123_587-KO_Z34C clone3
• B531 pSB1C3_oo1_CMV clone1
• B532 pSB1C3_oo1_CMV clone2
• B533 pSB1C3_001_CMV_ZEGFR:1907_VP2/3_HSPG-KO clone1
• B534 pSB1C3_001_CMV_ZEGFR:1907_VP2/3_HSPG-KO clone2
• B535 pSB1C3_001_VP2/3_ins_HSPG-KO_CMV_ZEGFR:1907 clone 1
• B536 pSB1C3_001_VP2/3_ins_HSPG-KO_CMV_ZEGFR:1907 clone 2
• B537 pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone1
• B538 pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone2
• B539 pBS1C3_VP2/3_capins_His clone1
• B540 pBS1C3_VP2/3_capins_His clone2
• B541 pSB1C3_ZEGFR:1907 _middlelinker_EGFP_His clone1
• B542 pSB1C3_ZEGFR:1907 _middlelinker_EGFP_His clone2

Mini-Prep was performed according to the standard protocol

• P644 pSB1C3_001_RC_IRCK_VP1-ko_HSPG-ko_P5tataless cl1 c= 151,0 ng/µl
• P645 pSB1C3_001_RC_IRCK_VP1-ko_HSPG-ko_P5tataless cl2 c= 115,8 ng/µl
• P646 pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1 c= 173,1 ng/µl
• P647 pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl2 c= 119,9 ng/µl
• P648 pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone1 c= 226,8 ng/µl
• P649 pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone2 c= 290,3 ng/µl
• P650 pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone3 c= 284,2 ng/µl
• P651 pSB1C3_001_CMV_VP123_587-KO_Z34C clone1 c= 283,8 ng/µl
• P652 pSB1C3_001_CMV_VP123_587-KO_Z34C clone2 c= 290,5 ng/µl
• P653 pSB1C3_001_CMV_VP123_587-KO_Z34C clone3 c= 251,7 ng/µl
• P654 pSB1C3_oo1_CMV clone1 c= 199,5 ng/µl
• P655 pSB1C3_oo1_CMV clone2 c= 192,7 ng/µl
• P656 pSB1C3_001_CMV_ZEGFR:1907_VP2/3_HSPG-KO clone1 c= 200,3 ng/µl
• P657 pSB1C3_001_CMV_ZEGFR:1907_VP2/3_HSPG-KO clone2 c= 325,0 ng/µl
• P658 pSB1C3_001_CMV_ZEGFR:1907_VP2/3_HSPG-KO clone3 c= 222,9 ng/µl
• P659 pSB1C3_001_CMV_ZEGFR:1907_VP2/3_HSPG-KO clone4 c= 201,3 ng/µl
• P660 pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone1 c= 267,1 ng/µl
• P661 pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone2 c= 193,1 ng/µl
• P662 pBS1C3_VP2/3_capins_His clone1 c= 209,1 ng/µl
• P663 pBS1C3_VP2/3_capins_His clone2 c= 252,5 ng/µl
• P664 pSB1C3_ZEGFR:1907 _middlelinker_EGFP_His clone1 c= 185,7 ng/µl
• P665 pSB1C3_ZEGFR:1907 _middlelinker_EGFP_His clone2 c= 192,0 ng/µl
• P666 pSB1C3_CFP c= 212,7 ng/µl

Test digestion of pSB1C3_ZEGFR:1907 _middlelinker_EGFP_His and mCherry iGEM 2008

Investigator: Jessica

Midiprep of pSB1C3_001_CMV_VP1/2/3_capins_587bla and pSB1C3_001_RC_IRCK_P5tataless clone1

Investigators: Chris W.

Midi-Preps of:
pSB1C3_001_CMV_VP1/2/3_capins_587bla =P667
pSB1C3_001_RC_IRCK_P5tataless clone1 =P668

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