Team:Humboldt Berlin/Notebook

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Notebook

Date Description
15.04.2020 - 30/08/2021 Drylabfirst introduction to use glycoproteins as surface marker/specificity marker, research to possible immune responses ⇒ lectins as final surface proteins
05/08/2020 - 26/03/2021 Drylab initial research to minicells (how to create them using the MinCDE system, advantages to normal Salmonella cells, virulence, purification)
16/11/2020 - 03/03/2021 Wetlabconstruction of ST14028 MvP103 sseC::aphT Para::hilA+ ΔminD::FRT strain
09/12/2020 succesfull insertion of ΔminD::fcf in EM11446
succesfullinsertionofDminD::FCFinEM11446
02/02/2021 - 29/05/21 Drylab research for an alternative way to create mini cells using the ftsZ gene (discontinued)
01/03/2021 - 09/10/2021 Wetlabconstruction of a plasmid for PlldR (BBa_K1847008) characterization in Salmonella Typhimurium
30/09/2021 successful cloning of pSB1C3-PlldR-gfp in Salmonella (EM7653)
successful cloning of pSB1C3-PlldR-GFP in EM7653
07/06/2021 Wetlab construction of mCherry and gfp expressing ΔminD::FRT strain for visualization of minicells
10/06/2021 - 24/06/2021 Wetlab visualization of minicells
11/06/2020 first mini cell purification results using Salmonella (EM7653), EM12053)
1st mini cell purification results using EM12052 and EM12053
15/06/2021- 15/08/2021 Drylab Design of pSB1C3-derived pKF01, the empty backbone that expresses thyA and lldR constitutively from the synthetic promoter Psyn-thyA. Thereupon, the plasmid pKF02 was designed in silico. pKF02 is supposed to express sicP-sptP-pep8 from the lactate-inducible promoter PlldR.
11/07/2021 Drylab introduction to the protocol written by Joyce Karlinsey and optimized for Salmonella Typhimurium LT2 in the lab of Dr. Kelly T. for allelic replacement using lambda-RED and tetRA replacement
20/07/2021 Drylab introduction to using agarpads for fluorescence microscopy
09/08/2021 - 26/09/2021 Wetlab visualization of Minicells II - DiSC and FM4-64
09/08/2021 second minicell purification results using Salmonella (EM7653)
Purification results using an optimized mini cell purification pre- and post-filtration in EM12052.
22/09/2021 purification results using an optimized mini cell purification pre- and post-filtration in Salmonella (EM7653)
Purification results using an optimized mini cell purification pre- and post-filtration in EM12052.
12/08/2021 Drylab introduction to galE deletion via tetRA insertion and replacement
12/08/2021 wetlab galE deletion via tetRA insertion and replacement
14/08/2021 - 09/10/2021 Wetlab cloning the pep8-Biobrick and PlldR-sicP-sptP-Biobrick into the pSB1A3 backbone
20/09/2021 succesfull insertion of PlldR-sicP-sptP into pSB1A3
succesfull insertion of PlldR-sicP-sptP into pSB1A3
14/08/2021 - 14/09/2021 Wetlab construction of ST14028 MvP103 sseC::aphT DthyA for pKF01 cloning
26/08/2021 successful replacement of ThyA-Gene with tetRA cassette
successful replacement of ThyA-Gene with TetRA cassette
14/08/2021 - 07/09/2021 Wetlab construction of DgalE mutants in ParaBAD::hilA strains
23/08/2021 successful insertion of the tetRA cassette within galE
insertion of tetRA within galE
07/09/2020 DgalE-clean was introduced at the right position 07/09/2021
Colony PCR - check if DgalE-clean was introduced at the right position
22/08/2021 - 26/09/2021 Wetlab visualization of Minicells II - DiSC and FM4-64
23/08/2021 successful insertion of the TetRA cassette within galE
25/08/2021 Drylab getting familiar with the DNA assembly - BioBrick NEB
24/09/21 - 26/09/21 Drylab design of the Codon optimized E_0840 part for Salmonella Typhimurium LT2
25/09/2021 - 26/09/2021 Wetlabminicell purification to determine minicell age distribution
26/09/2021 determine the minicell age distribution by quantifiying mini cell number based on OD values at given time points using Salmonella (EM7653)
Minicell purification to determine Minicell age distribution
25/09/2021 - 05/10/2021 Wetlabconstruction of plasmid for PlldR (BBa_K1847008) characterization in Salmonella Typhimurium
04/10/2021 successfull construction of pSB1C3-PlldR-gfp
27/09/2021 Wetlabgrowth curve of minicell producing strains
30/09/2021 - 09/10/2021 Wetlablife span/membrane integrity of minicells via Disc assay
Life span/Membrane integrity of minicells via Disc assay
01/10/21 Drylab design of PlldR-gfp (E_0840) part to test the inducible promoter
01/10/2021 - 16/10/2021 Wetlabassessment of production rate and size determination of minicells
11/10/2021 production timelapse of minicells in our ΔminD Salmonella Typhimurium strain
production timelapse of minicells in our ΔminD Salmonella typhimurium strain
09/10/2021 Wetlabcloning AIDA-rBC2LCN in pSB1C3
12/10/2021 - 21/10/2021 Wetlabcharacterization of the PlldR-gfp promoter in Salmonella Typhimurium
14/10/2021 pKF01-PlldR-gfp cloning succeeded

Date Description
April 2020 collecting modeling and implementation ideas
May 2021 modeling the metabolism rate as well as the lifespan of minicells, design of experiments to determine the lifespan (for example Proton Motive Force)
June 2021 outlines of population models for minicells by differential equations
July 2021 statistical modeling, assumption of log-normal distribution for the size of the minicells, binomial distribution for number of generated minicells
August/September 2021 Data analysis with CellProfiler, Fiji and Python
October 2021 Analysis of sedimentation rate from laboratory is coupled to modeling, improvement of population model

Date Description
Mar 2020 Working on the TargetTaxi Logo
Apr 2020 Announcement of "TargetTaxi" on Instagram and Facebook with the first Zoom-Team Photo
Oct 2020 Foundation of the PR Team: Philipp Kittler, Olyssa Sperling
Nov 2020 Working out a plan to explain our Lab Work
Dec 2020 Salmonella-as-SantaChristmas Post on Instagram
Jan 2021 Working on a TargetTaxi Flyer
Apr 2021 "The Minicell in a Nutshell"
May 2021 "Phase one: production ofminicells", "Phase two:secretion of proteins and adhesion to cancer cells", "Phase three: apoptosis of the cancer cell", “We are participating!” Announcement on Instagram
Jun 2021 New members in the PR Team: Vincent Fölsche and Martje Grensemann, Zoom-Meeting with iGEM Iiserb Bhopal from India about a Collaboration, Announcement that we are finally allowed to work in the lab, Zoom-Meeting with UBC iGEM from Vancouver, Canada, about our Collaborationon Salmonella Typhimurium
Jul 2021 Planning and shooting scenes for our Promotion Video
Aug 2021 The publishing of our Promotion Video, Meeting with Promega about Sponsorship Ideas
Sep 2021 Introduction of the PR Sub Team on Instagram and Facebook, Planning scenes and structure of our Presentation Video
Oct 2021 Collaboration Posts with UBC iGEM on Cancer and Salmonella, Shooting Scenes for our Presentation Video, Introduction of theLab and Modelling Sub Teams on Instagram and Facebook, Publishing of our Presentation Video

Date Description
Jan 2020 brainstorming who we could contact (biotech and pharmaceutical companies, foundations, crowdfunding maybe) and researching contact information
Feb 2020 making a list of things we should address during phone calls and making a template for emails to potential sponsors
Mar 2020 making first phone calls and sending emails
Oct 2020 deciding on different levels of rewards for sponsors based on how much money they give
Jan 2021 designing a flyer for sponsors, requiring Eppendorf’s and NEB's support
Feb 2021 requiring Roth’s support
Mar 2021 requiring IBA lifesciences' support
Mar 2021 requiring Promega's support
Sep 2021 requiring Microsynth Seqlab’s support
Oct 2021 requiring Dunn Labortechnik's, BioRender's and Gilson's support