Notebook HEARTBEAT

Welcome to the notebook of the HEARTBEAT (Heidelberg Artificial Transcription Factor Binding Sites Assembly and Engineering Tool) project. This notebook comprises the work on three sublanes: HEARTBEAT database (DB), HEARTBEAT graphical user interface (GUI) and HEARTBEAT fuzzy modeling (FN) as well as some additional work on logo as well as wiki design. Have fun!

Contents

8-3-2009

• got YFP/CFP-plasmids of Robert (Cellmembrane, ER, Cytosole)
• Joel pledged to give us further color contructs (Cellmembrane, Mitochondria, Nucleus)

8-4-2009

• transformation of all fluorescent proteins from upstairs (P.32-P.44 --> culture and miniprep tbd tomorrow)

8-5-2009

• transformation of all plasmids with targeted fluorescent proteins (P.32-P.43, P.44 still missing)
• made bacterial stocks of p32-43 and placed in -80°C freezer

8-6-2009

• Miniprep of p32, p33, p34, p35, p36, p37, p38, p39, p40, p41, p42, p43

8-7-2009

• Ordered primers for: GPI-attachement signal, Ras1b, YFP, CFP, GFP, mi

8-12-2009

• ordered primers for the mutagenesis PCR of the fluorescent proteins

8-14-2009

• two mutagenesis PCR's were done (p33g259a and p37a789g)

8-17-2009

• the other mutagenesis PCR's were done
• and all mutagenesis PCR products were transformed into DH5alpha bacterial cells

8-18-2009

• no colony because of problems with the kanamycin plates
• all mutagenesis PCR's were repeated
• started to test the ligation orientation of inserts into BBb plasmids:

- Digested p31 and a Jet insert with NheI and SpeI. -Ligated both together for 3 hrs at RT. (insert:vector= 1:1, 3:1, 1:3) -Transformed bacteria with ligate and plated them on Amp plates. -Repeated mutagenesis PCR again for (p33, p35, p37, p37-Ras, p41, p43)

8-19-2009

• Ligation worked for all 3 plates. Picked 6 colonies for minipreping from each plate for analysis.
• PCR amplification of each of GPI-attachement signal, EBFP alone, NLS alone and EBFP together with NLS.
• Ran EBFP/NLS amplicons on gel and the run seemed to work.

eBFP/NLS PCR amplification

• GPI-attachement signal was amplified overnight.

8-20-2009

• Restriction digestion of ligation products after minipreping.
• Ran PCR products for GPI on gel--> nothing!:(
• Restriction digestion showed only efficiency of 5% :( (only 1/18 digests showed insertion in the opposing direction)

8-21-2009

• new mutagenesis PCR (without kit)

8-22-2009

• Insert Amplification of mitoneet-eGFP by PCR

8-24-2009

• Restriction digest of mutagenized Plasmids (PstI) and analysis on gel

• Amplified inserts were gel-purificated

• What worked: eBFP, eBFP+NLS, eBFP_kozak, eBFP+NLS_kozak, eGFP, eGFP_kozak
• What didn't: NLS, NLS_kozak, eGFP+mitomeet, mitomeet, eGFP+mitomeet_kozak, mitomeet_kozak,

8-26-2009

BBBing of Insertsequences

• PCR of cherry, cherry_myrpalm, myrpalm, NLS with kozak Primers to amplify cherry_kozak, cherry_myrpalm_kozak, myrpalm_kozak, NLS_kozak

• Restriction with NheI and SpeI of localisationsequences and Flourophores, Restricted Plasmid was provided by Synthetic Promoter Group and digested with SAP
• Ligation with p31
• Transformation in DH5alpha with ligated Plasmids
• Outplating of Transformed cells on Amp-plates

8-27-2009

• Ligation and Transformation did not work (no colonies, except of two on the NLS )
• New PCR with flourophores and localisationsequences, to get higher amounts

• GEl purification of: eGFP, eGFP_kozak, eBFP, eBFP_NLS, eBFP_kozak, eBFP_NLS_kozak, NLS_kozak, cherry, cherry_myrpalm, myrpalm, cherry_kozak, cherry_myrpalm_kozak, myrpalm_kozak

8-28-2009

BBBing of Insertsequences2.0

• Restrictiondigest of flourophores and localisationsequences with SpeI and NheI (1 h, Buffer 2, BSA)
• Restrictiondigest of p49 with SpeI and NheI (1 h, Buffer 2, BSA) and SAP (30 min), purification
• Nanodrop of digest shows no DNA inside of the samples -- purification was maybe unsuccessful

8-29-2009

BBBing of Insertsequences2.1

• Restrictiondigest of flourophores and localisationsequences with SpeI and NheI (1 h, Buffer 2, BSA)
• Restrictiondigest of p49 with SpeI and NheI (1 h, Buffer 2, BSA) and SAP (30 min), purification

8-31-2009

BBBing of Insertsequences2.1 (part 2)

• Ligation of Insertsequences with restricted p49
• Transformation
• Outplating -> Wrong resistance