Team:TU Darmstadt/Notebook

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NOTEBOOK

See Methodic Protocols and Materials →

Labjournal 2016

Lab Weeks

1 (04/18 - 04/24)	2 (04/25 - 05/01)	3 (05/02 - 05/08)	4 (05/09 - 05/15)	5 (05/16 - 05/22)
6 (05/23 - 05/29)	7 (05/30 - 06/05)	8 (06/06 - 06/12)	9 (06/13 - 06/19)	10 (06/20 - 06/26)
11 (06/27 - 07/03)	12 (07/04 - 07/10)	13 (07/11 - 07/17)	14 (07/18 - 07/24)	15 (07/25 - 07/31)
16 (08/01 - 08/07)	17 (08/08 - 08/14)	18 (08/15 - 08/21)	19 (08/22 - 08/28)	20 (08/29 - 09/04)
21 (09/05 - 09/11)	22 (09/12 - 09/18)	23 (09/19 - 09/25)	24 (09/26 - 10/02)	25 (10/03 - 10/09)
26 (10/10 - 10/16)	27 (10/17 - 10/23)

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 experienced students offered classes like a snapgene workshop (March 4), how to use scifinder (March 12) and a cloning strategies workshop (March 17).

Lab Week 1: April 18 - April 24		This week, we organised the lab work before the actual work started.
		Robotics Meeting with the people interested in the technical part of the project, discussing the task and the possible ideas.
		We met on Tuesday with the whole team.

Lab Week 2: April 25 - Mai 1		This week, we organised the lab work before the actual work started.
		Robotics Decision who will participate in the project and which direction we chose. 4 people form the team and we do some research. Idea is to redesign a 3D printer
		We went to the local Weinlagenwanderung to get out, drink some quality wine and enjoy some great views.

Lab Week 3: May 2 - May 8		This week, we organised the lab work before the actual work started.
		Robotics Meeting and making a team commitment.
		We met on Tuesday with the whole team.

Lab Week 4: May 9 - May 15		Killswitch BBa_K1976020: The OMT (O-Methyl-L-tyrosine) tRNA synthetase was synthesized without ribosome binding site by IDT. PCR was performed with prefix and suffix primers. BBa_K1976024: The OMT tRNA was synthesized without ribosome binding site by IDT. PCR was performed with prefix and suffix primers.
		Robotics Brainstorm the possible ideas, discussing the found approaches. Need of more research
		We met on Tuesday with the whole team.

Lab Week 5: May 16 - May 22		Killswitch BBa_K1976020: The OMT tRNA synthetase and psB1C3 vector were cut with the restriction enzymes EcoRI and PstI. The two restriction products were ligated by standard T4‑Ligation protocol. The ligation product was transformed into E. coli Top 10 via heatshock. BBa_K1976024: The OMT tRNA and psB1C3 vector were cut with the restriction enzymes EcoRI and PstI. The two restriction products were ligated by standard T4‑Ligation protocol. The ligation product was transformed into E. coli Top 10 via heatshock.
		Robotics Deciding the components and the programming language. Details still open.
		We met on Tuesday with the whole team.

Lab Week 6: May 23 - May 29		miniColicin The BBa_K1976048 was made by performing a PCR on a product synthesized by IDT, using the primers prefix right and suffix right and digesting it using EcoRI and PstI. 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. Killswitch Colicin E2 was synthesized including B0034 ribosome binding site (RBS) by IDT. It was cut with the restriction enzymes EcoRI and PstI, than ligated by standard T4‑Ligation protocol with a psB1C3 vector which was also cut with EcoRI and PstI but the ligation failed. BBa_K1976021: We added the B0034 RBS to OMT tRNA synthetase via PCR and the primer S_FW-R.
		Robotics 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 for the materials for the syringe pump and one will think about the detection with an optical detection system.
		We met on Tuesday with the whole team.

Lab Week 7: May 30 - June 5		miniColicin The brick BBa_K1976049 was created by performing PCR on a product synthesized by IDT, using the primers prefix right and suffix right and digesting it using EcoRI and PstI. 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 Killswitch BBa_K1976021: The PCR product OMT tRNA synthetase with RBS was cut with the restriction enzymes EcoRI and PstI, than ligated by standard T4‑Ligation protocol with a psB1C3 vector which was also cut with EcoRI and PstI. The ligation product was transformed into E. coli Top 10 via heatshock.
		We continued with the work from the last weeks
		We met on Tuesday with the whole team.

Lab Week 8: June 6 - June 12		Killswitch BBa_K1976022: We took the BioBrick BBa_J23101 and psB1C3 cut them with the restriction enzymes EcoRI and PstI, than ligated by standard T4‑Ligation protocol and transformed the product into E. coli Top 10 via heatshock. BBa_K1976028: The Colicin E2 immunity protein was synthesized including B0034 ribosome binding site and amber codon by IDT. It was cut with the restriction enzymes EcoRI and PstI, than ligated by standard T4‑Ligation protocol with a psB1C3 vector which was also cut with EcoRI and PstI. The ligation product was transformed into E. coli Top 10 via heatshock. BBa_K1976026: To eliminate the B0034 RBS in the immunity protein we used the primers I-FW and I_REV and PCR method. We cut the product and psB1C3 with EcoRI and PstI, than ligated by standard T4‑Ligation protocol and transformed the product into E. coli Top 10 via heatshock.
		Robotics 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.
		We met on Tuesday with the whole team.

Lab Week 9: June 13 - June 19		Killswitch BBa_K1976022: After plasmid preparation BBa_K1976021 was cut with XbaI and PstI und the psB1C3 with the constitutive promoter BBa_J23101was cut with SpeI and PstI than ligated by standard T4‑Ligation protocol.
		We continued with the work from the last weeks.
		The group had a weekend in the Austrian Alps, where we decided on our Human Practices project.

Lab Week 10: June 20 - June 26		Killswitch BBa_K1976022: The ligation product was transformed into E. coli Top 10 via heatshock.
		Robotics 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.
		We met on Tuesday with the whole team.

Lab Week 11: June 27 - July 3		Metabolic burden Promoter 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 transformation. The strength of the three promoters was then compared by observing the fluorescence of the GFP. Killswitch BBa_K1976033: We cut T7 promotor BBa_K525998 and psB1A3 with EcoRI and PstI, than ligated by standard T4‑Ligation protocol and transformed the product into E. coli Top 10 via heatshock.
		Robotics 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
		We met on Tuesday with the whole team.

Lab Week 12: July 4 - July 10		Killswitch BBa_K1976022: The ligation product was transformed into E. coli BL21 via heatshock for protein expression and SDS PAGE.
		Robotics 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
		We met on Tuesday with the whole team.

Lab Week 13: July 11 - July 17		Killswitch BBa_K1976032: We used the same cloning strategy as with BBa_K1976022. BBa_K1976028 was cut with XbaI and PstI und the psB1C3 with the constitutive promoter BBa_J23101was cut with SpeI and PstI than ligated by standard T4‑Ligation protocol.
		Robotics We will build 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.
		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: July 18 - July 24		miniColicin The BBa_K1976050 was made by performing a PCR on a product synthesized by IDT, using the primers M_RBS_DNAse_FW and suffix right. Following that another PCR was performed using prefix right and suffix right and digesting it using EcoRI and PstI. 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. E. coli Top10 were then transformed with the ligated plasmid. Following that a colony PCR was performed to check for the proper band height. Killswitch BBa_K1976032: The product of ligation was transformed into E. coli Top 10 via heatshock. BBa_K1976033: As in BBa_K1976032, BBa_K1976028 was cut with XbaI and PstI, the psB1A3 with T7 Promoter was cut with SpeI and PstI than ligated by standard T4‑Ligation protocol. The ligation product was transformed into E. coli Top 10 via heatshock.
		Robotics 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.
		Laura stays for another week.

Lab Week 15: July 25 - July 31		miniColicin The BBa_K1976051 was made by performing a PCR on a product synthesized by IDT, using the primers M_RBS_DNAse_FW and suffix right. Following that another PCR was performed using prefix right and suffix right and digesting it using EcoRI and PstI. 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. E. coli Top10 were then transformed with the ligated plasmid. Following that a colony PCR was performed to check for the proper band height. Killswitch BBa_K1976024: Due to the fact that the OMT tRNA we tryed to assemble with psB1C3 multiple times had a point mutation, we decided to design the mutagenese primers T_FW-M, T_REV-M-B (for blunt ends) and T_REV-M-S (for sticky ends) and perfomed a mutagenese PCR.
		We discussed some problems and continued to work on the robot.
		We met on Tuesday with the whole team.

Lab Week 16: August 1 - August 7		Killswitch BBa_K1976024: Unfortunately our mutagenese experiment ended up in a deletion mutation, so we decided to continue working with the point mutation OMT tRNA. BBa_K1976022 was cut with SpeI and PstI, OMT tRNA was cut with XbaI and PstI, than ligated by standard T4‑Ligation protocol. The ligation product was transformed into E. coli Top 10 via heatshock.
		Robotics 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
		Our team went to the annual iGEM Germany Meet-Up in Marburg and exchanged ideas with the other german teams.

Lab Week 17: August 8 - August 14		Reporter System BBa_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. Killswitch BBa_K1976029: To eliminate the amber coden in BBa_K1976028 we used the primers I_REV-M, I_REV-M-B (for blunt ends) and I_REV-M-S (for sticky ends) and the PCR method. Both versions worked and the product was cut with EcoRI and PstI, than ligated with psB1C3 cut with EcoRI and PstI by standard T4‑Ligation protocol. The ligation product was transformed into E. coli Top 10 via heatshock. BBa_K1976030: Also the colicin E2 immunity protein without the amber coden from BBa_K1976029 was cut with XbaI and PstI, and psB1C3 with constitutive generator J23101 was cut with SpeI and PstI, than ligated by standard T4‑Ligation protocol. The ligation product was transformed into E. coli Top 10 via heatshock and plasmid preparation as performed.
		Robotics We are playing with the stepper motors and waiting for some final parts for the x-y-axis.
		The project coordination team planned a Pokémon-Style event for team bonding and we played some games and had great fun!

Lab Week 18: August 15 - August 21		Reporter System BBa_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. Killswitch BBa_K1976031: Like BBa_K1976030 the immunity protein was assembled with psB1A3 with Promotor T7 and transformated in E. coli Top 10 via heatshock.
		Robotics 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 are working on the GUI and the Qt software
		We met on Tuesday with the whole team.

Lab Week 19: August 22 - August 28		Reporter System BBa_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. Killswitch BBa_K1976031: After plasmid preparation BBa_K1976031 was transformated in E. coli BL21 via heatshock for protein expression and SDS PAGE.
		We assembled some parts of the robot and spend some time getting used to the programs we use.
		We met on Tuesday with the whole team.

Lab Week 20: August 29 - September 4		Reporter System BBa_K1976002: Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones. Metabolic burden BBa_K1976001: The BioBrick BBa_I11020 was synthezised with a LVA‑tag and with B0034 as a ribosome binding site by IDT. Killswitch After multiple failures to assemble Colicin E2 with psB1C3 or psB1A3 we tryed Circular Polymerase Extension Cloning (CPEC) with overhang primers.
		We mostly spend our time on the computer.
		We met on Tuesday with the whole team.

Lab Week 21: September 5 - September 11		Reporter System BBa_K1976002: Isolated Plasmid DNA Sequence was verified by Sanger sequencing. Metabolic burden BBa_K1976001: The LVA‑tag was deleted by PCR with the primer Del_LVA_fw and Del_LVA_rev. Killswitch BBa_K1976033: After plasmid preparation BBa_K1976033 was transformated in E. coli BL21 via heatshock for protein expression and SDS PAGE.
		Robotics 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.
		Our team team visited Evonik and was granted the opportunity to see how they work.

Lab Week 22: September 12 - September 18		Reporter System BBa_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 burden BBa_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. Killswitch Due tot he fact that the OMT tRNA had a point mutation which could not be changed via mutagenese PCR, the orthogonal pair was synthesized by IDT. It was cut with the restriction enzymes EcoRI and PstI, than ligated by standard T4‑Ligation protocol with a psB1C3 or psB1K3 vector which was also cut with EcoRI and PstI but the ligation failed several times.
		Robotics 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.
		We met on Tuesday with the whole team.

Lab Week 23: September 19 - September 25		Reporter System BBa_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 burden BBa_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. Killswitch We also tryed to assemble the synthesized orthogonal pair via CPEC, but like Colicin E2 it failed.
		Robotics The GUI is in 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.
		We met on Tuesday with the whole team.

Lab Week 24: September 26 - October 2		Reporter System BBa_K1976003: Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones . miniColicin BBa_K1976054 was made by digesting Bba_K1976048 with Xba1 and Pst1. Also BBa_K525998 was digested with EcoRI and PstI. Both were ligated into pSB1A3. The ligation Top10 E. coli were then transformed with this construct. A colony PCR was performed to check for the right band height. Lastly the construct was digested with EcoRI and PstI and ligated in pSB1C3, which was digested the same way. Metabolic burden BBa_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.
		Robotics 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 Killswitch BBa_K1976031: The DNA construct including T7 Promotor and psB1C3 vector were cut with the restriction enzymes EcoRI and PstI. The two restriction products were ligated by standard T4‑Ligation protocol. The ligation product was transformed into E. coli Top 10 via heatshock. BBa_K1976033: The DNA construct including T7 Promotor and psB1C3 vector were cut with the restriction enzymes EcoRI and PstI. The two restriction products were ligated by standard T4‑Ligation protocol. The ligation product was transformed into E. coli Top 10 via heatshock.
		We met on Tuesday with the whole team.

Lab Week 25: October 3 - October 9		Reporter System BBa_K1976003: The isolated Plasmid DNA was verified by Sanger sequencing. miniColicin BBa_K1976055 was made by digesting Bba_K1976049 with Xba1 and Pst1. Also BBa_K525998 was digested with EcoRI and PstI. Both were ligated into pSB1A3. The ligation Top10 E. coli were then transformed with this construct. A colony PCR was performed to check for the right band height. Lastly the construct was digested with EcoRI and PstI and ligated in pSB1C3, which was digested the same way. Metabolic burden BBa_K1976000: The LVA‑tag was added by PCR with the overhang primer LVA_GFP_fw and LVA_GFP_rev.
		Robotics 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. Killswitch BBa_K1976031: Plasmid preparation as performed. BBa_K1976033: Plasmid preparation as performed.
		We met on Tuesday with the whole team.

Lab Week 26: October 10 - October 16		Reporter System BBa_K1976003: Subsequently the psB1A3 vector containing brick was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4‑Ligase. miniColicin The BBa_K1976052 was made by performing a PCR on Bba_K1976050 using Prim-0.36-FW and suffix right and digesting it using EcoRI and XbaI. 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 EcoRI and PstI and dephosphorylated, using our standard protocol for the antarctic phosphatase. Following that using the standard T4 protocol, these fragments were ligated together. E. coli Top10 were then transformed with the ligated plasmid. Following that a colony PCR was performed to check for the proper band height. The BBa_K1976053 was made by performing a PCR on BBa_K1976050 using Prim-0.36-FW and suffix right and digesting it using EcoRI and XbaI. 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 EcoRI and PstI and dephosphorylated, using our standard protocol for the antarctic phosphatase. Following that using the standard T4 protocol, these fragments were ligated together. E. coli Top10 were then transformed with the ligated plasmid. Following that a colony PCR was performed to check for the proper band height. Metabolic burden BBa_K1976000: 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.
		Robotics 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.
		We met on Tuesday with the whole team.

Lab Week 27: October 17 - October 23		Metabolic burden The cells with the genomic integrated B0034 construct were made chemically competent and then transformed with the naringenin-sensor BBaa_K1497020 from iGEM 2014. The metabolic burden was measured via microplate reader. The cells were induced with naringenin.
		Robotics Time is running and we are working on the last details. We are exited for the Jamboree.
		We met on Tuesday with the whole team.

iGEM Technische Universität Darmstadt

We thank our sponsors, especially:

Email: igem@bio.tu-darmstadt.de | Design: Viktoria Schuster