03.09.2012

Main topic: Task distribution for the week. Upon discussion with the Aptamer Team (Ali and Maryam) and Praveen, the following tasks were scheduled to be done during this week:

• Do a chart comparing the prices, delivering times, characteristics for the PDGF that the different companies offer. (Aptamer Group)
• Contact Ralf Seidel (Seidel’s group has stuff from Sigma, so they might have some discount or partnership) or Sigma-Aldridge directly, as they have Human PDGF BB in their stock, and ask for a sponsorship or a discount for the team. (Ali is working on it). (Aptamer Group)
• Order the optimal PDGF (AS SOON AS POSSIBLE!!-> The delivery times are around 2 weeks!!!). (Aptamer Group)
• Ask Michael Schlierf if an spectrophotometric assay might be the best technique for determining the reaction kinetics of the aptamer in labeled and unlabeled conditions. If it is; start right away as soon as the PDGF is here!!! If not, then ask him what he would suggest (FRET?)(Aptamer Group)
• Contact Dominik to ask about the new chol modified sequences that Yixin sent and confirm with Alex and Santiago if they do not overlap with the strands in the origami structure. (GUVs and Origami Group)
• In case they overlap, they have to be immediately ordered from a company (no time for synthetizing them) using sequences that do not overlap for sure. (GUVs Group)
• Ask Dominik and Ralf about the Quantum Dots usage, assemble new structures with QDs and start running the respective experiments; LSM for the GUVs and Gel Shift for the Origami. (GUVs and Origami Group)
• TEM image all the different DNA Origami constructs that were assembled to determine the best conditions for the origami and for negative staining. (Origami Group)
• Determine the Layout and colors of the wiki webpage. Discuss the logo. (Santiago)
• Meet Marino Zerial at 3:00pm tomorrow (05.09.2012) to discuss about possible applications for the project. IMPORTANT: The feedback we get from Marino should be used to write the ABSTRACT of the project!!!!!! The more or less final draft has to be done the latest on Friday, so that next week on Monday we can meet with our supervisors for final corrections!!!!!! REMEMBER!!! ---> The abstract has to be send before Friday 14.09.2012 to shawn.douglas@wyss.harvard.edu. (Everyone).
• Next Meeting on Friday 07.09.2012 before Beer Hour in Biotec (16:00). Results/progress evaluation!!

31.08.2012

Summary

• The biotin labelled oligos arrived.
• The aptamers and complements arrived.
• The labelling was done till today as the biotin labelled oligos arrived till wednesday
• Ask Yixin Zhang about the cholesterol labelled oligos->How does it work? could we do it? also discuss TEG spacer with him.

REMARKS/COMMENTS:

• Option given by svea->Polystyrene beads (1um) if QDs don't work.
• Keep in mid Articles in German for enterprises.

IMPORTANT TASKS: To order (Everybody should be aware):

• Catcher complementary + chol @ 3'->To connect LUVs
• Anchor complementary + chol @ 3'->To connect the DNA origami with the GUVs IMPORTANT!!!!!

To do:

• Clarify the procedure for chol labelled oligos and the TEG spacer -> Yixin Zhang Cost and time are key!!!! (Praveen)
• Talk with the technician (or someone) that know about the process. To be done on monday. (Praveen)
• Get PDGF. Santiago (me) will insit with MP Biomedicals. But please-> Look for other companies and harrass them if it's necessary!!(Ali and Maryam)
• Start aptamer experiments right away. Characterize aptamer and test the locks->Does labelling affect/change function? (Ali and Maryam)
• Do GUV-QD exps. during this week-> Attach complementary stands to GUVs and QDs and do microscopy (Varsha, Praveen)
• Assemble new constructs and do Origami-QD exps-> Gel shift and TEM (Alex, Santiago)
• Advance with the wiki!! -> Define a solid layout (Santiago)

20.08.2012

Fahrgarten Review and remarks from 17.08.2012

• Check the size of the construct vs. the GUV’s size. Do they fit?, What about QDs
• Where aptamers ordered with BHQs?
• General stuff about Visa procedures->Transport, bus/No telephones or handpack
• T-shirts and logo should be designed probably using Dresden skyline in a comic version (siluette)

17.08.2012

General description of the aptamer.

• Send a mail to Shawn Douglas to check the consistency of the ordered aptamers.
• Is it the same to label the 3’ with Cy3 and 5’ with BHQ as doing the other way around? The company sponsoring the aptamers suggests labeling on 3’ with the dye.
• Around the last ~15 bases of the aptamer are the ones that actually react with the protein during the aptamer conformation change.
• Check where we can buy the PDGF. How much does it cost? I checked and it costs 254 euros for 25ug and 7741 for 10mg
• Check how the aptamer labeling works?
• Check the kinetics of the aptamer -> How quick is the aptamer -> Measure speed of aptamer, having quencher and fluorophore.
• Fluorophore and quencher are around 1~1.5 nm.
• Can actually one make the sequence longer without altering the functioning of the aptamer?
• NOTE: Ask if they actually desisted of doing the aptamer labeling because of its difficulty
• Ralph suggest that we test opening and close by binding. Why bothering with the specific lock closing, if the proof of concept is binding?
• QD to prove that 655nm QD->15nm in case that the LUVs doesn’t work QD
• Gel shifting upon the binding of QDs -> Nice way of probably testing the binding of QDs .
• Additionally CCS (Cross Cor. Spec) can be performed also.
• Probably we don’t need both locks to prove the concept.
• Kinetics of the aptamer is ok to show. Prove and recook.
• Risky strategy suggested by Michael . Order the 4 oligos (aptamers and complements) and add to additional aptamers with the 3’
• PAGE gel with the labeled oligo and see difference between hybridized and non hybridized structures.
• Heating up the sample during the assembly process
• Run a simple hybridization process Heat and cool, observe fluorescence changes in time. Leave it in the Fluor. Spectrometer -> We can do it in B CUBE, they

DNA Origami

• Run the gel with QDs for the origami. Biotin attaching to the 5’ ends of the catcher strands and connected to QDs. Begin with a central strand -> check for a shift in a gel. Test hybridization. Order additional linkers through Sygma, the linkers also Biotin labeled to test the hybridization. Basically QD replace the LUV.
• Santiago’ll do negative staining with Susanne Kretschmar (ask for the proper preparation time witout biasing her).

Summary/Discussion with supervisors

• Ask Douglas about the “mysterious” TTs and the Mg++ solution.
• Run a control for GUV-> Have a GUV coated with strands and try to bind the QDs with the complementary bps. If the QDs work we can go for the origami.
• Next step would be to check the specific attachment of the DNA origami with the GUVs. Probably
• The proportion of biotinated oligos should be slightly over 1:1, though 1:1 is ok.
• Purify out (flush out) the linkers so that the single ones do not interfere by binding to the LUVs
• Ask Dominik to order QDs and Biotin.
• Order oligos and Biotins and run experiments both in GUV and DNA origami side!!!
• Contact the following company for asking about sponsorship and to order the PDGF MP Biomedicals GermanyToll Free Tel: 00800 7777 9999 Tel: 0800-426 67 337 Toll Free Fax: 0800-629 67 337 Email: custserv.de@mpbio.com
• Prepare the aptamer sequences and order them:
• Run some kinetics on the aptamers alone-> Nice to show in results.

13.08.2012

Start-> 17:30
Origami (Alex):

• Cost 1160 euros, 7560 bp.
• Origami descrition see presentation and dropbox for extra details
• Question: Why the locks are not symmetric
• Effective radio of strands in the origami: 2.25nm, extra 0.25nm given by possible repulsion between strands.
• 45nmx37.35x40.5-44nm
• Two versions->guided, not guided
• Edge staples. Possible correction issues.
• Versions tried->Open shell, closed shell
• Separation by gel extraction->Doesn’t it damage the structure???
• IDEA: PCR Purification to separate single oligos from structure.
• Staining->which technique use Douglas for staining?
• Different protocols for staining 3 drops of water, 1 drop. Which one is the best??
• Is DNA interacting with the grid?? Maybe as it is laying It can change its structure and affect the results. Check with Douglas paper.
• Results->Good: Distinguishable regular spots, Bad: No differentiation between open and closed versions
• Errors in staining->probably during centrifugation and taking too much time.
• We should do a gel with different buffers, i.e, different concentrations of Mg(2+).
• Probable clash between Mg concentrations for DNA and GUV experiments.
• Maybe adding long catcher strands could help the structure to stay open.
• Schedule staining session with Suzanne Kretschmar and TEM session with Thomas Kurth.
• Question: How is DNA labeled?? Could we use QDots??

Santiago part/Wiki and Origami:

• Origami->Santiago has to be aware of every single detail of the DNA origami, know the sequences, structure of the origami, length, locks, guide strands.
• Gockan designed the webpage for I-GEM. He knows a lot about javascripting and Photoshop. Contact him!! Use I-Gem web pages as a measuring stick for webpage. BIOMOD ones are not that good.

Finances:

• Who’s gonna do the finances? -> Varsha
• Tell to juliane, avoid any confusion for her.

GUVs:

• Problem-> Still clustering.
• Possible solutions-> Change buffer concentration and length of the linker
• LSM meeting tomorrow at 10:00->Praveen, Varsha and Mari are attending
• Aptamer group-> Ali should check the kinetic coefficients and know them by heart, not just saying that they’re on the paper.
• We have to know things to order from the sponsorship of the companies-> we have to do it fast; if it’s too late companies could be reluctant to give them.
• Start Downloading the pictures made with the LSM for safety issues

04.08.2012

GUVs meeting Experiment schedule sketch:

• LUVS and GUVs with or without Mg
• LUVS and GUVs with col oligos
• GUVs had red and green fluorescence. LUVs are dots, possible fluorescence contamination.
• Check GUVs with a normal microscope Last experiment:
• Electroformation
• Oligo coupling
• After 3 days all the GUVs and LUVs had clustered now matter is there was oligos or not. Maybe there was a problem with the linker.

Start on Monday:

• Microscope shifts begin at 9:30am. Usually are booked till 8:00pm.
• IMPORTANT: The cholesterol adding step to GUVs has to be done overnight.
• Dominik said that entropy of the system might destabilize the structure.
• Book the microscope overday and overnight
• Int 4’s without cholesterol are gonna be tested with linker and only GUVs. Linker and labeled oligos at the same time.
• How much time should the oligos be left for hybridizing? It’s around 25 mins, but we could let them for

Experiments
Experiment 1:

	LUVs+GUVs –Mg 2+	LUVs+GUVs –Mg 2+
LUVs+GUVs –Mg 2+ int4 int30 linker	LUVs+GUVs +Mg 2+ int4 int30 linker	LUVs+GUVs –Mg 2+ int4 int30

Experiment 2:

LUVs+GUVs –Mg 2+	LUVs+GUVs +Mg 2+	LUVs –Mg 2+	LUVs -Mg 2+ int4
LUVs+GUVs –Mg 2+ int4 int30	LUVs+GUVs +Mg 2+ int4 int30	LUVs+GUVs +Mg 2+ int4

Experiment 3:

Alexa int 4/linker/int 30/GUVs	Alexa int 4/linker/int 30/GUVs	LUVs/int 4/Linker	LUVs/int 4
LUVs	GUVs/int 30/Linker/LUVs/int 4/-Mg+2	GUVs/int 30/LUVs/int 4/-Mg+2	GUVs/LUVs/-Mg+2
GUVs/LUVs/int4	GUVs/int 30/Linker/LUVs/int 4/+Mg+2	GUVs/int 30/LUVs/int4/+Mg+2	GUVs/LUVs/+Mg+2

• Microscopes: 510(noon) and 780(afternoon)
• Booking System

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30.07.2012

1. Thomas will contact TU Dresden again to get the Money they promised as well as GSK Foundation
2. Meeting this week with Juliane Hoth (Thursday or Friday: 8 am):
   1. Varsha and Praveen, Santiago will go with Thomas to get an overview over funding.
   2. Meeting with Supervisors beginning of next week (Aga will organize: maybe next monday):
   3. Money issue (what is process so far, contact of companies? Zeiss, Nikkon, JPK,….)
   4. Who is able to join us in the US?
   5. We have some companies who support us with Material, what kind of Material should we get, who needs what? Aptamer group: Needs to work this week, exams are over!
3. Questions to be answered this week about the aptamer group:
   1. Which Aptamer do we use? (Which Oligosequence?)
   2. How do we evaluate the opening of the Aptamer (Arrange a meeting with Michael: FRET!!)
   3. Experimental design – (PP – Presentation next week – merge with Origami group)
   4. What kind of FRET dyes do we need (Quencher – Donor), where can we buy it and how much, how is the labeling procedure (talk to Dominik)?
4. GUV group:
   1. Order Oligos
   2. Use Oligos and stain them with Fluorophor
   3. Dominik has stains to label Oligos at 3-Prime (the one who is used for the linkage to the GUV…just without the cholesterol),
   4. A meeting needs to be arranged to get introduction to Microscopy! (Arrange meeting this week to be maybe next week or the week after: ask Aleksander (Olec) who is responsible)
   5. AIM: Work on your own, without supervisors look over the shoulders…soon!!
   6. Procedure: aleksander expects that the procedure will take a whole day.
   7. Sequence: Electroformation: at 9 am, Microscopy late in the afternoon.
   8. Check with the Fluorophores, Problem might be the Linker, couple Cholesterol with new Oligo or try everything out with the Structure itself,
   9. Maybe one can do TEM with the GUV coupled Origami
5. Biolympics who takes care?
   • Include some other class mates and Svea (Varsha will take care!)
6. Wiki:
   • Schedule: How to see the work until the abstract submission in September until end of the week, everybody!!
7. Origami:
   • Somebody should get an introductory course to work with TEM (additionally to Alex)
8. Break:
   • We can not afford to stop working due to vacation! We will modify the editgrid where everybody can put in when they work.
9. Meeting with Opitz:
   • He maybe can pay a flight, but has to talk to other directors at fraunhofer. He will get back to us in the middle of August.
10. T-Shirts:
    • Somebody has to design the T-Shirts (Middle of September…: Karen will design a CUTE Dinosaur^^),
11. Application of our system to sell the story in the presentation. Meeting with Marino Zerial next week (Praveen),
12. How many want to have a Matlab license?
    • Santiago, Praveen, Varsha, Aga (one B CUBE computer: 5 Licenses: Santiago organizes it).
13. Meeting with Transinsight:
    • They will probably support us with 200 € and he (CEO: Michael Alvers) will support us with marketing hints (we should send him the presentation and present in front of him to get hints and shape the basic idea out, make it understandable and provoke questions); He will get back to us after the decision.
14. VISA Photos: Thomas will send around where he did them – he knows visa requirements of USA,
    ##Questionnaire, Letter from BIOTEC that you are studying here, BIOTEC Transcript of records needed please contact Anne / Martina immediately
15. Thomas will make Flowcharts for What happens if a company gives us money? What happens if I want to order sth?
16. Regular Meetings: Mondays: 5:30 pm; Fridays: 4 pm (before beer Hour, Update of the week!!)