Team:Freiburg Bioware/testpage

6. Labday 03.05.2010

Theoretical cloning

Investigators: Adrian, Hanna, Bea, Patrick, Chris W.

There are several matters to do in theoretical cloning. The modularization/modification of the stratagene plasmids and the enzymes. :

1. Cap-Gen:

• delete the PstI-restriction site
• insert the antibody fragment (targeting): sequence? in which part of VP1? (Patrick)
• add prefix & suffix
• disable binding of Heparan Sulphat Proteoglycan

2. Rep-Gen:

• delete EcoRI (2x) and PstI (2x)

3. ITRs:

• NotI and PstI (in sequence?) flank the ITRs: its the question if we can/must delete them?
• where exactly starts and ends the sequence (Patrick)?
• it should be checked up if there is a possibility to use all of the three ITRs.
• we blasted the ITR (left) of the MCS vektor (Stratagene). we got a 92% "Coverage" with the AAV2 genom (to 100%). from the alignment (Stratagene ITR with ITR of the AAV2 genom) we got:
  

You can see, that the ITR sequence beginns 4 bp after the PstI restriction site. thereupon we did a secundary structure analyse (www.dinamelt.bioinfo.rpi.edu), with the following structurewe got:
Media:Freiburg10_AAV2ITR(left)nachBlast.pdf
conclusion: the big loop of the secondary structure dosent change if we delete the PstI restriction site (cf. with other uploadet secondary structures), it should be considered that we can't add random bp that could affect the secundary structure.
To do: which bp, which sequence could/can be insertet? cf. the genome of AAV2

4. MCS:

• replacement through the iGEM-MCS
• where is the beginning and ending of the MCS in the Vector? ß-globin function and sequence (search for literature and patents, which are denoted in the AAV-Helper-Free-System Manual)
• in this context it would be also important to find out where the beta-Globulin-Intron exactly starts or rather we can cut out the MCS.

5. enzymes:

• Thymidinkinase: find informations. TK30 (Bea), SR39 (Hanna)
• Cytosindeaminase: find informations (Adrian)

in addition we downloadet, addet and anotated the sequence of the pHelper plasmid of stratagene in Geneious (see "Constructs").

insert the antibody fragment (targeting): sequence? in which part of VP1? (Patrick)

7. Labday 07.05.2010

Protocolls

Investigators: Anissa, Kerstin

• adaption and extension of the standart prorcolls (Cloning for Pro's)
• we startet to create a protokoll-mask for the praktical-cloning (short version for labwork)

8. Labday 10.05.2010

Stock solutions

Investigators: Adrian, Chris W., Chris L., Bea, Achim, Patrick, Hanna, (Sven)

The following stock solutions were prepared:
1. Antibiotics:

• Ampicillin: 2 g Ampicillin were dissolved in 20 mL ethanol (70%), filled into 2 mL tubes and stored at -20°C.
• Chloramphenicol: 0.5 g Chloramphenicol were dissolved in 20 mL ethanol (70%), filled into 2 mL tubes and stored at the -20°C.
• Kanamycin: 1 g Kanamycin was dissolved in 20 mL multipore-H2O, sterilized by filtration and filled into 2 mL tubes and stored at the -20°C.
• Tetracyclin: 0.5 g Tetracyclin were dissolved in 20 mL ethanol (70%) and filled into 2 mL tubes. The tubes were wrapped with aluminium foil (light sensitive!) and stored at -20°C.

2. ITPG solution (1 M):

• ~ 4.766 g ITPG were dissolved in 20 mL multipore-H2O, sterilized by filtration, filled in 2 mL tubes and stored at -20°C.

3. DYT (5 litres)

• 80 g Bactotrypton, 50 g Bactoyeast, 25 g NaCl were weight out.
• 2 L multipore-H2O were added
• after mixing, multipore-H2O was added -> endvolume 5 litres
• medium was filled into flask and was autoclaved

4. Glycerol:

• Glycerol was filled into a flask and was then autoclaved

To do: register at Mr. Gene!!!

9. Labday 17.05.2010

β-Globin

Investigators: Bea, Chris W., Patrick, Hanna (and instructors)

We blasted the sequence of the β-globin and additional nukleotides „vorne und hintendran“. We found only 70% coverage with the human β-globin-intron add. some parts of the exon 3. For this reason we think that the pAAV_MCS annotation of the β-Globin-intron of Stratagene is too generously.
In addition the alignment of the human ß-globin showed that only parts of the intron 2 (5’) and exon 3 are integratet into the vector. We assume that the informations from Stratagene are not total correctly ( intron flanket by the splicedonors and the acceptor-sequence). We blasted the sequence between the CMV-promoter and the origin beginning of the ß-globin intron. We found a 98,8% coverage with a synthetic CMV-promoterconstruct ( 1 nucleotide difference). Literature: „Diverse plasmid DNA vectors by directed molecular evolution of cytomegalovirus promoters. (Wright A. et al.)“
→The question arises if we can omit the ß-globin (because the exact function is unknown). For this we should contact various Companys (GeneArt, DNA2.0, Mr. Gene,…) to get more information. In addition we could test the expression with and without ß-globin.

10. Labortag 18.05.2010

pCMV_mVenus_YFP

Investigator: Bea, Chris W., Patrick, Hanna, Anissa, Kerstin, Adrian (und Instructors)

Theoretical cloning with Geneious of pCMV-MCS + pGA14_mVenus_YFP --> pCMV_mVenus_YFP

• Enzyme set: RFC 25 (iGEM)
• digest pGA14_mVenus_YFP (insert) with XbaI and PstI: mVenus_YFP_cut_XbaI+PstI
• digest pCMV_MCS (vector) with XbaI and PstI: pCMV_MCS_cut_XbaI+PstI
• ligate mVenus_YFP_cut_XbaI+PstI with pCMV_MCS_cut_XbaI+PstI
  

11. Labortag 19.05.2010

Cloning of pCMV-MCS + pGA14_mVenus_YFP --> <b>pCMV_mVenus_YFP

Investigators: Adrian, Bea, Chris W., Hanna, Patrick

Digestion

• plasmid: insert: pGA14_mVenus_YFP; number: P1 production date: ____ origin: ____
• plasmid: vector: pCMV_MCS; number: P2 production date: ____ origin: ____
• new vector name: pCMV_mVenus_YFP
  
• buffer used:3 ; Restriction-enzymes used: Enzyme XbaI (no. Lab:___) ; Enzyme PstI(no.Lab:___)
• DNA concentration (vector): 375 ng/µl ; DNA concentration (insert): 476 ng/µl

components	V (pGA_mVenus_YFP)/ µl	V(pCMV_MCS) / µl
DNA	4	2,7
BSA (10x)	2	2
Buffer 3 (10x)	2	2
Enzyme: XbaI (no.Lab:___)	1,5	1,5
Enzyme: PstI (no.Lab:___)	1	1
H2O	9,5	10,8
Total volume	20	20

• Incubation: 1 h at 37°C

1% Agarose gel and Gel extraction

• prepare 1% agarose gel, run gel for 45 minutes(119 V)
• cut out insert and vector
• perform gel extraction following standard protocol provided by Qiagen

Ligation

• Measure DNA-concentration with Nanodrop
• c(mVenus_YFP) = 16,8 ng/µL
• c(pCMV_MCS) = 22,8 ng/µL
• Calculation of volume needed for ligation:
• c(mVenus_YFP) = 3,66 µL
• c(pCMV_MCS) = 5,34 µL

Transformation

• Transformation has been followed the standard protocol Media:Freiburg10_Cloning Protocol.pdf

12. Labortag 20.05.2010

Picking clones

Investigators: Adrian, Bea

Clones were picked according to the standard protocol.
• 3 approaches from each plate

13. Labortag 21.05.2010: CMV-Promoter

Theoretical cloning: (Volker, Hanna)

• The CMV promoter (mainly the regulatory region) was further characterized: For this purpose U.S. patent no. 5,385,839 was used. Media:Freiburg10_Patent_US5385839A.pdf
• Further on the CMV promoter sequence was blasted. The results delivered a 98% query coverage with the "Human herpesvirus 5 strain Toledo, complete genome" (accession no.: GU937742.1). Interestingly the maximal identity was just 99%. This can be explained due to a nucleotide deletion and a C-T transition (red circles), which were also marked in Geneious.

To do: find "+1"-location (transcription start); which transcription factors bind to the regulatory region of the CMV promoter?

LB medium was prepared: (Patrick and Chris W.)

• 10 g Bacto-Tryptone, 5 g Bacto-Yeast, 10 g NaCl were mixed in 500 mL milipore-H2O.
• Volume was adjusted to 1 L with milipore-H2O.
• 100 mL flasks were each filled with 50 mL medium.
• 0.75 g agar was added to each flask.
• LB was sterilized by autoclaving and is now stored at room temperature.

Plasmid Mini-Prep according to the standard protocol

• investigator: Adrian, Kira, Anna, Chris W., Patrick
• Measure DNA-concentration with Nanodrop

14. Labortag 25.05.2010

Cloning of pCMV_mVenus_YFP + pAAV_MCS

Investigators: Kira, Anna, Volker, Jessica