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

User =Biomod12
PWD= !bio12mod#

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!!)

02.07.2012

BIOMOD Meeting

1. Long Night of science:
   1. Cardboard for the DNA Origami -> ask Birte or in the lab; Velcro for the GuVs , what are the smaller balls? Ping-Pong? Or tennis? Or sth. even bigger? Alex bought polystyrene balls, We need straws for the Origami-Oligos, We need double sided tape to attach the Velcro (the Velcro already has glue on one side…). -> we will put it together on Thursday. We need some candys ;)
   2. Alex will buy Velcro and Polystyrene spheres,
   3. Posters: We will make the photos on Wednesday at 5 pm!!!, also already for the homepage… . We have booked the printer on Thursday afternoon. Could all go over the posters and take a look please (look in Dropbox). Plese wear for the photoshooting: Jeans and T-Shirts with intense colours,
   4. Shifts of the Long Night of Science: 3 people for 2 ½ h. Be here already at 5 pm, Agata will make a doodle for the shifts.
   5. We get 5 T-Shirts altogether and vouchers for a meal at the canteen, two bottles (one water, one apple juice) for the station -> so you should bring sth. for yourself I guess this is not enough for the whole evening
2. Update about the lab work:
   1. The GUV group makes quite good progess in learning the techniques… Exam break for 2-3 weeks now…
3. Visa: Please inform yourself about the length of the Visa process as soon as possible so we can estimate the time you need for the whole process ##We need to talk to the supervisors who will accompany us to Boston? Harvard University will pay for the accommodation…so they need to know how many people are going so they can arrange the rooms. ##Visa process: please don´t apply so far but gather the information what you need to get a visa and collect all the information (e.g. letter from BIOTEC that you are a student here….). Suggestion: the Visa application should be done then in the beginning of August as soon as we know how much money we approx. get from the companies (how supportive they are…) and so we know for how many people we can pay the flight.

28.06.2012

BIOMOD Meeting:

• We need to rearrange the groups, because the GUV group (Aptamer tests also included so far…) has lots of work to do and so far there are only molbioengineers in the group…Praveen, Agata, Karen…who wants to join this group from NanoBios? We want to establish a new group who is working with Aptamers + do the FRET experiments in B CUBE with Michael Schlierf.
• The GUV group works with Aleksander and he requires the people to be very well prepared, the paper has to be read, the calculations be done, the experiments planned…

  DNA Origami	GUV's	Aptamer
  Design & the structure (mainly Alex) assembly of structure will be lots of pipetting work…	Agata, Praveen, Karen, Varsha, Santiago	Mari + (Praveen, Aga or Karen)
  TEM (Alex and Ali for sample preparation)

• Alex probably could need some help, for the TEM sample preparation -> Ali will help him and already joins him tomorrow (29.6.) for the TEM measurement (9-12),
• Varsha is going to the Long Night of Science meeting tomorrow for introduction at 10 am, in CRTD seminar room one
• The posters should be translated until Sunday evening, Thomas will take care that they are printed in time + reserve the printer time….(done)
• Thomas will give the names of the participating people to Birte for the badges (done).
• What kind of 3D model should we do for the long night to entertain the people and to explain our project?
  • Build a box, with straws colour coded and attach GUV to GUV…. We should make the model entertaining…what kind of materials, please think a little bit more about the structure. We should get velcro, Big GUV with parts covered with sandwich velcros, LUVs with another Velcro but cannot attach to the sandwich, we have the aptamer ball (which mimics our aptamer key system) which takes away the top of the sandwich Velcro so the LUV with the fitting Velcro can attach. Question: where do we get the Velcro? For making the Origami structure we could use polystyrene->Alex will ask/look in the lab.
• Everybody is expected to be in CRTD for the Long Night of Science…at least for a few hours, It starts at 6 officially, but we should be there at 4 or 5.
• Funding:
  • We have more or less three accounts over which we can order things (BIOTEC, CRTD, DIGS-BB)-> we should not loose the overview. Therefore as soon as you order please state it in the Editgrid from which account you want to take it and contact the responsible person (BIOTEC: Juliane Hoth, CRTD: Ilona Kreher, DIGS-BB: Dana Jaster)
  • Alex will be the responsible person for ordering over the DIGS-BB account-> Thomas will write an email to DIGS-BB (done)
  • Unfortunately the Saxon Ministry of Science and Arts has no money for student projects…
  • The letters have been put together by Thomas today and sent….let´s see what replies we will get.

21.06.2012

• We have lab space ( 2 benches in B CUBE), we can also hang out there because they have a desk for us. Michael Schlierf already contacted a company with the funding. We should talk to the people of the Microscopy / TEM facility in BIOTEC, because these people know the companies very well and buy expensive equipments there… .
• GUV diameter is around 10 µm. In Schlierf´s lab they do LUVs with 100 nm -> which are stable
• Flat surface of the Origami after we induce the opening and get rid of the lid. Maybe have a little helix bundle as spacer between the surface so we have no sterical hindrance with the LUV,
• IDEA: If there would be a problem with the sterical hindrance of a stiff, flat Origami sheet we could add staples and linker strands which are longer and shorter in the middle.
• Tracking the opening of the DNA Origami: Using a quencher and a donor system which changes the fluorescence after conformational change, You can do up two colors. How would we stain Origami? Put in some fluorophores on staple strands, (10 fluorophores on the Origami, put them far away). There are Aptamers which can bind to ATP (we don´t expect to have ATP in our system) which seem to be pretty stable, they had to heat it up to 37°C, GC sequences which are usually more stable -> thermodynamically
• IDT: Online programme (Integrated DNA Technologies) is a quite good software to predict secondary structures … .
• Creation of LUVs with 100 nm can be in the range of 100 nm +- 20 nm;
• Problem of one LUV binding to several Origamis on a big GUV -> probably not an issue because of the curvature of the LUV,
• In the origami structure we maybe have to consider to use supporting strands which we get rid after the successful assembly of the structure.
• To find a proper aptamer we should go through the papers, and also look for papers who cite this paper (ATP Aptamer paper, which Michael gave to us),
• We want to use two Origami structures but there could be some problems:
• Because the dimensions need to fit really exactly, maybe we can use instead a single strand hinge and use the principle also used for the nanorobot (science
• Maybe the Origami is too complex, so we maybe should have a backup…just to attach an Origami to the GUV,
• Parallel approaches: Play with the Aptamer in solution if it binds correctly (characterizing it in experiments, identify it). How much does an aptamer with the fluorophores cost? Fluorophores order of 200 – 300 € , Order the DNA functionalized with a group and then attach the fluorophore using protocols already established in B CUBE.
• Bending of Origami is not 100 % controllable
• Papers about Aptamer:
  • “Structure-Switching Signaling Aptamers” – Razvan Nutiu and Yingfu Li (McMaster University)
  • “A DNA-Protein Nanoengine for “On-Demand” Release and Release and Precise Delivery of Molecules” – Razvan Nutiu and Yingfu Li

14.06.2012

Meeting with Dr. Zhang and discussion about the project People: Alexander, Karen, Svea, Praveen, Ali, Santiago, Varsha, Agata, Thomas, Dr. Zhang Time: 17:30

1. Alexander presented the Steps for the experiment-> Find presentation in the googlegroup
   1. We need three different cholesterol labelled oligos
   2. 9 connections to anchor the Origami on the outside of the GUV
   3. Ask Dominik to use the origami structure anchored to a guv (already in press in the paper);
   4. B CUBE has a pool of 30 different staples/oligos coupled to cholesterol;
   5. One cholesterol is added to one oligonucleotide: the big challenge is the solubility, with more cholesterols added
   6. There is no good method to purify it because the cholesterols would cluster together and the oligos stay outside, the cholesterol is added at the 3’ end; so the staples hanging out must be also complementary at the 3’ end.
   7. Origami coupling to the GUV: combining one anchor strand with a staple

*Discussion about getting rid of one of the lids, so we would not have the problem with a possible sterical hindrance, could be possible with different lock systems at the correct/proper places *How many origamis? Coat the GUV with LUVs, so nearly coat the origami completely *How much time is needed to synthetize the cholesterol coupled oligos? *In B-cube (Dr. Zhang) they have already a lot of coupled oligos (in storage) and for the coupling you really need not many… *Problem with connecting GUVs for the control (because no cholesterol labelled complementary strands): oligos can form hairpin structures so it should be possible; have a linker oligo *We have to think about a partly double stranded structure to stabilize it *Meeting tomorrow at noon: at B-cube (to discuss with Svea and Michael Schlierf about the control experiment and the attachment of the two GUVs ) *Karen will ask Dominik Kauert if he has any oligos left (stock solutions) which are labelled with cholesterol and which one for the sequence