iGEM 2016 — Team TU_Darmstadt

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# Team:TU Darmstadt/Notebook

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![iGEM TU Darmstadt 2016](https://static.igem.org/mediawiki/2016/8/83/T--TU_Darmstadt--titel.png)

![menu](https://static.igem.org/mediawiki/2016/c/c9/T--TU_Darmstadt--zahnrad.png)

* [Achievements](https://2016.igem.org/Team%3ATU_Darmstadt/Achievements)
* [Wet Lab](https://2016.igem.org/Team%3ATU_Darmstadt/Lab)
* [Parts](https://2016.igem.org/Team%3ATU_Darmstadt/Parts)
* [Robotics](https://2016.igem.org/Team%3ATU_Darmstadt/Hardware)
* [Modeling](https://2016.igem.org/Team%3ATU_Darmstadt/Model)
* [Human Practices](https://2016.igem.org/Team%3ATU_Darmstadt/Human_Practices)
* [Collaborations](https://2016.igem.org/Team%3ATU_Darmstadt/Collaborations)
* [Notebook](https://2016.igem.org/Team%3ATU_Darmstadt/Notebook)
* [Results](https://2016.igem.org/Team%3ATU_Darmstadt/Results)
* [Team](https://2016.igem.org/Team%3ATU_Darmstadt/Team)

![teamphoto](https://static.igem.org/mediawiki/2016/8/86/T--TU_Darmstadt--team.jpg)

# NOTEBOOK

[See Methodic Protocols and Materials →](https://2016.igem.org/Team%3ATU_Darmstadt/Notebook/Methods)

## Labjournal 2016

Lab Weeks

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| --- | --- | --- | --- | --- |
| [**1**  (04/18 - 04/24)](#lw1) | [**2**  (04/25 - 05/01)](#lw2) | [**3**  (05/02 - 05/08)](#lw3) | [**4**  (05/09 - 05/15)](#lw4) | [**5**  (05/16 - 05/22)](#lw5) |
| [**6**  (05/23 - 05/29)](#lw6) | [**7**  (05/30 - 06/05)](#lw7) | [**8**  (06/06 - 06/12)](#lw8) | [**9**  (06/13 - 06/19)](#lw9) | [**10**  (06/20 - 06/26)](#lw10) |
| [**11**  (06/27 - 07/03)](#lw11) | [**12**  (07/04 - 07/10)](#lw12) | [**13**  (07/11 - 07/17)](#lw13) | [**14**  (07/18 - 07/24)](#lw14) | [**15**  (07/25 - 07/31)](#lw15) |
| [**16**  (08/01 - 08/07)](#lw16) | [**17**  (08/08 - 08/14)](#lw17) | [**18**  (08/15 - 08/21)](#lw18) | [**19**  (08/22 - 08/28)](#lw19) | [**20**  (08/29 - 09/04)](#lw20) |
| [**21**  (09/05 - 09/11)](#lw21) | [**22**  (09/12 - 09/18)](#lw22) | [**23**  (09/19 - 09/25)](#lw23) | [**24**  (09/26 - 10/02)](#lw24) | [**25**  (10/03 - 10/09)](#lw25) |
| [**26**  (10/10 - 10/16)](#lw26) | [**27**  (10/17 - 10/23)](#lw27) |  |  |  |

For half a year we were working in the wet lab  ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png)  , developed a pipetting robot  ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) and simulated the molecular dynamics of our proteins  ![](https://static.igem.org/mediawiki/2016/7/78/T--TU_Darmstadt--iconMod.png)  .
As well we would like to highlight certain social events  ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) 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).

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| Lab Week 1: April 18 - April 24 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | This week, we organised the lab work before the actual work started. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsMeeting with the people interested in the technical part of the project, discussing the task and the possible ideas. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 2: April 25 - Mai 1 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | This week, we organised the lab work before the actual work started. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsDecision 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 |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We went to the local Weinlagenwanderung to get out, drink some quality wine and enjoy some great views. |

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| Lab Week 3: May 2 - May 8 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | This week, we organised the lab work before the actual work started. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsMeeting and making a team commitment. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 4: May 9 - May 15 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsBrainstorm the possible ideas, discussing the found approaches. Need of more research |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 5: May 16 - May 22 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsDeciding the components and the programming language. Details still open. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 6: May 23 - May 29 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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.  KillswitchColicin 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsMade 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 7: May 30 - June 5 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | miniColicinThe 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. |

|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | We continued with the work from the last weeks |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 8: June 6 - June 12 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 9: June 13 - June 19 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | We continued with the work from the last weeks. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | The group had a weekend in the Austrian Alps, where we decided on our Human Practices project. |

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| Lab Week 10: June 20 - June 26 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | Killswitch**BBa\_K1976022:** The ligation product was transformed into *E. coli* Top 10 via heatshock. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsCADs 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 11: June 27 - July 3 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | Metabolic burdenPromoter test (J23109, J23101 and J23115):  pSB3K3‑BBa\_E0240 was cut with the restriction enzymes*XbaI* 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsThe 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 |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 12: July 4 - July 10 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | Killswitch**BBa\_K1976022:** The ligation product was transformed into *E. coli* BL21 via heatshock for protein expression and SDS PAGE. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe 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 |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team.  | |

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| Lab Week 13: July 11 - July 17 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | 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. |

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| Lab Week 14: July 18 - July 24 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | Laura stays for another week. |

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| Lab Week 15: July 25 - July 31 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | We discussed some problems and continued to work on the robot. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 16: August 1 - August 7 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe 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 |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | Our team went to the annual iGEM Germany Meet-Up in Marburg and exchanged ideas with the other german teams. |

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| Lab Week 17: August 8 - August 14 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe are playing with the stepper motors and waiting for some final parts for the x-y-axis. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | The project coordination team planned a Pokémon-Style event for team bonding and we played some games and had great fun! |

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| Lab Week 18: August 15 - August 21 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | Roboticsx-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 |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 19: August 22 - August 28 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | We assembled some parts of the robot and spend some time getting used to the programs we use. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 20: August 29 - September 4 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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.  KillswitchAfter multiple failures to assemble Colicin E2 with psB1C3 or psB1A3 we tryed Circular Polymerase Extension Cloning (CPEC) with overhang primers. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | We mostly spend our time on the computer. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 21: September 5 - September 11 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsMounted 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | Our team team visited Evonik and was granted the opportunity to see how they work. |

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| Lab Week 22: September 12 - September 18 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. KillswitchDue 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 23: September 19 - September 25 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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.  KillswitchWe also tryed to assemble the synthesized orthogonal pair via CPEC, but like Colicin E2 it failed. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsThe 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 24: September 26 - October 2 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsMunich 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 25: October 3 - October 9 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 26: October 10 - October 16 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsWe 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. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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| Lab Week 27: October 17 - October 23 | ![](https://static.igem.org/mediawiki/2016/b/b5/T--TU_Darmstadt--iconLab3.png) | Metabolic burdenThe 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. |
|  | ![](https://static.igem.org/mediawiki/2016/7/7c/T--TU_Darmstadt--iconTech.png) | RoboticsTime is running and we are working on the last details. We are exited for the Jamboree. |
|  | ![](https://static.igem.org/mediawiki/2016/c/ce/T--TU_Darmstadt--iconSocial.png) | We met on Tuesday with the whole team. |

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[iGEM Technische Universität Darmstadt](https://2016.igem.org/Team%3ATU_Darmstadt)

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We thank our [sponsors](https://2016.igem.org/Team%3ATU_Darmstadt/Team/Acknowledgements), especially:

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