Difference between revisions of "Team:TU Darmstadt/Notebook"
| Line 497: | Line 497: | ||
<b>BBa_K1976003:</b><br> | <b>BBa_K1976003:</b><br> | ||
Subsequently the psB1A3 vector containing brick was cut with <i>EcoRI</i> and <i>PstI</i> and ligated into a pSB1C3 vector using T4‑Ligase.<br> | Subsequently the psB1A3 vector containing brick was cut with <i>EcoRI</i> and <i>PstI</i> and ligated into a pSB1C3 vector using T4‑Ligase.<br> | ||
| + | <h6>miniColicin</h6> | ||
| + | |||
| + | <p>The BBa_K1976052 was made by performing a PCR on Bba_K1976050 using <i>Prim-0.36-FW</i> and <i>suffix right</i> and digesting it using <i>EcoR1</i> and <i>Xba1</i>. Also Bba_K1976029 was amplified by PCR using <i>Prim-0.72-FW</i> and <i>suffix right</i>. Following that both were ligated into the backbone pSB1C3 which was digested using <i>EcoR1</i> and <i>Pst1</i> and dephosphorylated, using our standard protocol for the antarctic phosphatase. Following that using the standard T4 protocol, these fragments were ligated together.<br> | ||
| + | E. coli Top10 were then transformed with the ligated plasmid. Following that a colony PCR was performed to check for the proper band height.</p><br> | ||
| + | <br> | ||
| + | <p>The BBa_K1976053 was made by performing a PCR on Bba_K1976050 using <i>Prim-0.36-FW</i> and <i>suffix right</i> and digesting it using <i>EcoR1</i> and <i>Xba1</i>. Also Bba_K1976029 was amplified by PCR using <i>Prim-0.72-FW</i> and <i>suffix right</i>. Following that both were ligated into the backbone pSB1C3 which was digested using <i>EcoR1</i> and <i>Pst1</i> and dephosphorylated, using our standard protocol for the antarctic phosphatase. Following that using the standard T4 protocol, these fragments were ligated together.<br> | ||
| + | E. coli Top10 were then transformed with the ligated plasmid. Following that a colony PCR was performed to check for the proper band height.</p> | ||
| + | |||
<h6>Metabolic burden</h6> | <h6>Metabolic burden</h6> | ||
Revision as of 22:56, 19 October 2016
NOTEBOOK
Labjournal 2016
For half a year we were working in the wet lab 
, developed a pipetting robot 
and simulated the molecular dynamics of our proteins 
.
As well we would like to highlight certain social events 
which helped us to further improve our team spirit.
Our iGEM year started with weekly team meetings on tuesdays in January.
On February 27 and 28 we officially iniated the project with our Kickoff-Event in which teams have been formed and weekly teamleader meetings were set.
To teach the new members all the needed basics for working in the lab and genetic engineering in general, the advisors offered classes like a snapgene workshop (March 4), how to use scifinder (March 12) and a cloning strategies workshop (March 17).
Lab Week 1: |
|
Hier könnte Ihr labjournal stehen! |
|
RoboticsMeeting with the people interested in the technical part of the project, discussing the task and the possible ideas. | |
|
modeling | |
|
social |
Lab Week 2: |
|
Hier könnte Ihr labjournal stehen! |
|
RoboticsDecision who will participate in the project and which direction we chose. 4 people forming the team and we do some research. Idea is to redesign a 3D printer | |
|
modeling | |
|
We went to the local Weinlagenwanderung to get out, drink some quality wine and enjoy some great views. |
Lab Week 3: |
|
Hier könnte Ihr labjournal stehen! |
|
RoboticsMeeting and making a team commitment | |
|
modeling | |
|
social |
Lab Week 4: |
|
Hier könnte Ihr labjournal stehen! |
|
RoboticsBrainstorm the possible ideas, discussing the found approaches. Need of more research | |
|
modeling | |
|
social |
Lab Week 5: |
|
Hier könnte Ihr labjournal stehen! |
|
Deciding the components and the programming language. Details still open. | |
|
modeling | |
|
social |
Lab Week 6: |
|
miniColicinThe BBa_K1976048 was made by performing a PCR on a product synthesized by IDT, using the primers prefix right and suffix rightand digesting it using EcoR1 and Pst1. Following that it was ligated into the backbone pSB1C3 which was digested the same way and dephosphorylated, using our standard protocol for the antarctic phosphatase. Following that using the standard T4 protocol, these fragments were ligated together. Lastly E. Coli Top10 have been transformed with the ligation |
|
Made decision about the chassis, an Ultimaker2 clone. Pumping system will be a syringe pump from hackaday.io Two will look for the needed materials for the chassis, one the materials for the syringe pump and one will think about the detection system with an optic. | |
|
modeling | |
|
social |
Lab Week 7: |
|
The BBa_K1976049 was made by performing a PCR on a product synthesized by IDT, using the primers prefix right and suffix rightand digesting it using EcoR1 and Pst1. Following that it was ligated into the backbone pSB1C3 which was digested the same way and dephosphorylated, using our standard protocol for the antarctic phosphatase. Following that using the standard T4 protocol, these fragments were ligated together. Lastly E. Coli Top10 have been transformed with the ligation |
|
robotics | |
|
modeling | |
|
social |
Lab Week 8: |
|
Hier könnte Ihr labjournal stehen! |
|
We start to design the required CADs and learn the CAD software to do so. One of us is still doing some research about the optical detection system and how to do it. | |
|
modeling | |
|
social |
Lab Week 9: |
|
Hier könnte Ihr labjournal stehen! |
|
robotics | |
|
modeling | |
|
The group had a weekend in the Austrian Alps, where we decided on our Human Practices project. |
Lab Week 10: |
|
Hier könnte Ihr labjournal stehen! |
|
CADs are ready and the bill of materials is finished with the supplier information. We order the parts and talk to our workshop if they can make the needed adjustments. Our optics is chosen to be a Raspberry Pi NoIR Camera and we are using the OpenCV package to do the detection work. | |
|
modeling | |
|
social |
Lab Week 11: |
|
Metabolic burdenPromoter test (J23109, J23101 and J23115):pSB3K3‑BBa_E0240 was cut with the restriction enzymesXbaI and PstI. J61002 with the promoter J23109, J23101 or J23115 was each cut with SpeI and PstI and then ligated with the cut BBa_E0240 construct. The ligation product was transformed into E. coli Top 10 via heatshock. The strength of the three promoters was then compared by observing the fluorescence of the GFP. |
|
The ordered materials arrived and we started assembling the chassis. We talked to the workshop and they prepare some of the parts for us. One of our team members is preparing the OpenCV software and getting familiar with the camera | |
|
modeling | |
|
social |
Lab Week 12: |
|
Hier könnte Ihr labjournal stehen! |
|
We have some problems assembling the chassis. Some parts need to be adapted. We like to have as much active measuring area as possible. The first 3D printed parts for the syringe pump are ready and we need to redesign some parts. The optics require IR light from underneath. Next week we are discussing the details of the construction | |
|
modeling | |
|
Lab Week 13: |
|
Hier könnte Ihr labjournal stehen! |
|
We will built a lightbox wit IR LEDs. To assure a homogeneous IR light we will use a diffuser plate illuminated from the sides. One team member prepares the technical drawing. | |
|
modeling | |
|
A guest from Milan arrives to get an impression of how iGEM works and help out in our lab - her name is Laura. She wants to introduce iGEM to her university as well and build a team there. |
Lab Week 14: |
|
Hier könnte Ihr labjournal stehen! |
|
We are working on the chassis, the syringe pump and the optics. We have first object detections from the camera and the main parts of the chassis are assembled. | |
|
modeling | |
|
Laura stays for another week. |
Lab Week 15: |
|
Hier könnte Ihr labjournal stehen! |
|
robotics | |
|
modeling | |
|
social |
Lab Week 16: |
|
Hier könnte Ihr labjournal stehen! |
|
We are waiting for the workshop to finish parts for the lightbox and the z-axis. We are working on the detection of rectangles and getting familiar with the Marlin software | |
|
modeling | |
|
Our team went to the annual iGEM Germany Meet-Up in Marburg and exchanged ideas with the other German teams. |
Lab Week 17: |
|
Reporter SystemBBa_K1976002:The mVenus-LVA-gene was synthesized by idt and amplified through PCR using the SP-amplify-fw-Rep1 (fw) and SP-amplify-rev-Rep2 (REV) oligonucleotides. |
|
We are playing with the stepper motors and waiting for some final parts for the x-y-axis. | |
|
modeling | |
|
The project coordiantion team planned a Pokémon-Style Event for team bonding and we played some games and had great fun! |
Lab Week 18: |
|
Reporter SystemBBa_K1976002:The PCR product was verified by agarose gel electrophoresis and subsequently purified. The mVenus amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. |
|
x-y-axis is mounted but we still need to adjust everything so it is moving smoothly The z-axis need a holdering made out of aluminum blocks we have to order. In that time we working on the GUI and the Qt software | |
|
modeling | |
|
social |
Lab Week 19: |
|
Reporter SystemBBa_K1976002:The ligation product was transformed into E.coli Top 10 by heat shock transformation and the cells were spread out on a Chloramphenicol agar plate. |
|
robotics | |
|
modeling | |
|
social |
Lab Week 20: |
|
Reporter SystemBBa_K1976002:Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones. Metabolic burdenBBa_K1976001:The BioBrick BBa_I11020 was synthezised with a LVA‑tag and with B0034 as a ribosome binding site by IDT. |
|
robotics | |
|
modeling | |
|
social |
Lab Week 21: |
|
Reporter SystemBBa_K1976002:Isolated Plasmid DNA Sequence was verified by Sanger Sequencing. Metabolic burdenBBa_K1976001:The LVA‑tag was deleted by PCR with the primer Del_LVA_fw and Del_LVA_rev. |
|
Mounted the z-axis and tuned the x-y axis. The optical software is nearly ready to use and we like to test it next week. We started connecting the electronics to an ATX power supply. The Raspberry Pi is set up and ready to use. We still have some problems with the wireless connection. | |
|
modeling | |
|
Our team team visited Evonik and was granted the opportunity to see how they work. |
Lab Week 22: |
|
Reporter SystemBBa_K1976003:The mVenus-LVA-pSB1C3 plasmid was cut with EcoRI and PstI and ligated into a T7-RBS-pSB1A3 plasmid which was cut with SpeI and PstI. Metabolic burdenBBa_K1976000:The BioBrick BBa_E0240 was cut with the restriction enzymes XbaI and PstI. BBa_J61002 was cut with SpeI and PstI. The two restriction products were ligated by standard T4‑Ligation protocol. BBa_K1976001: B0034‑BBa_I11020 was cut with XbaI and PstI. BBa_J23101 was cut with SpeI and PstI. Both fragments were ligated using the T4‑Ligase by standard protocol. |
|
We installed the LEDs, relays and drivers for the LEDs. Sometimes they are not working properly but it seems to be a problem of the software, one is focusing on that task. X-y-Axis and z-axis movement works. We are installing the endstops to assure the steppers motors are stopping at the end of the movement. | |
|
modeling | |
|
social |
Lab Week 23: |
|
Reporter SystemBBa_K1976003:The ligation product was transformed into E.coli Top 10 by heat shock transformation and the cells were spread out on a Ampicillin agar plate. Metabolic burdenBBa_K1976000:After plasmid preparation, the plasmid was cut with XbaI and PstI. We had the attp‑site BBa_11023 flanked by the two additional bidirectional transcription terminators BBa_1001 synthesized by IDT. The attp‑site of BBa_11023 was not the original λ‑attp‑site. Instead the sequence corresponded to the attP2‑site used in the Gateway ® cloning system. The construct was cut with EcoRI and SpeI. The pSB1C3 backbone was cut with EcoRI and PstI. Together with the previously cut BBa_J61002‑BBa_E0240 construct the synthesized BBa_1001‑BBa_11023‑BBa_1001 construct was ligated in the cut pSB1C3. BBa_K1976001: The ligation product was transformed into E. coli Top 10 via heatshock. |
|
The GUI is on principle ready to use. Our RAMPS board is maybe broken and we buy another one. The syringe pump was tested and it works like expected. | |
|
modeling | |
|
social |
Lab Week 24: |
|
Reporter SystemBBa_K1976003:Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones . Metabolic burdenBBa_K1976000:The attP2‑site was mutated to λ‑attp via Quick‑Change‑PCR, using the following primers: QC_attp_fw and QC_attp_rev (for more details see our primerlist). BBa_K1976001: To check whether BBa_I11020 was expressed properly a SDS‑Page was made. |
|
Munich sent us their syringe pump. We are now printing the additional parts and like to test it. Still working on the electronics and one LED strip needs to be soldered again. We installed our lightbox and we can clearly see the objects and have target vectors which we are using for our auto tracking later | |
|
modeling | |
|
social |
Lab Week 25: |
|
Reporter SystemBBa_K1976003:The isolated Plasmid DNA was verified by Sanger sequencing. Metabolic burdenBBa_K1976000:The LVA‑tag was added by PCR with the overhang primer LVA_GFP_fw and LVA_GFP_rev. |
|
We assembled the Munich syringe pump and tested it. It works good and we have new ideas for our system. We gave them feedback for their design. Our robot is working but we need to fix some bugs. Also we have to adjust some parameters. | |
|
modeling | |
|
social |
Lab Week 26: |
|
Reporter SystemBBa_K1976003:Subsequently the psB1A3 vector containing brick was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4‑Ligase. miniColicinThe BBa_K1976052 was made by performing a PCR on Bba_K1976050 using Prim-0.36-FW and suffix right and digesting it using EcoR1 and Xba1. Also Bba_K1976029 was amplified by PCR using Prim-0.72-FW and suffix right. Following that both were ligated into the backbone pSB1C3 which was digested using EcoR1 and Pst1 and dephosphorylated, using our standard protocol for the antarctic phosphatase. Following that using the standard T4 protocol, these fragments were ligated together. The BBa_K1976053 was made by performing a PCR on Bba_K1976050 using Prim-0.36-FW and suffix right and digesting it using EcoR1 and Xba1. Also Bba_K1976029 was amplified by PCR using Prim-0.72-FW and suffix right. Following that both were ligated into the backbone pSB1C3 which was digested using EcoR1 and Pst1 and dephosphorylated, using our standard protocol for the antarctic phosphatase. Following that using the standard T4 protocol, these fragments were ligated together. Metabolic burdenBBa_K1976000:For plasmid curing the final plasmid construct and the low copy backbone pSB4A5 were cut with EcoRI and PstI and then ligated. BBa_K1976001: For plasmid curing the final plasmid construct and the low copy vector pSB4K5 were cut with EcoRI and PstI and then ligated. BBa_K1976000 and BBa_K1976001 were cotransformed into E. coli Top 10. To check whether the genomic integration was successful a cPCR with attb_fw and VR primers was made. |
|
We are testing the functionality of the robot. Unfortunately we still have no mVenus to work with, however we can do some test with our light detection. We encapsulated the robot and can control it via network. We are finalizing the robot | |
|
modeling | |
|
social |
Lab Week 27: |
|
Metabolic burdenPlasmid curing could not be performed due to lack of time. |
|
Time is running and we are working on the last details. We are exited for the Jamboree. | |
|
modeling | |
|
social |
