14.06. – 20.06.
Getting started
- This week we can finally get started in the lab and spend the week organising the lab, preparing buffers, medium
- Chemocompetent E.coli DH5α cells and Heat shock transformation to test them (Paul)

21.06. – 27.06.
Gene syntheses arrive
- The gene Synthesis for our signaling cascade in bacteria and tobacco arrived, so we started our attempts to amplificate them (Louisa)
- Testing the competent cells through heat shock transformation (Paul, Lennart)
-Creating glycerin-stocks

28.06. – 04.07.
Starting reporters
-Sequencing of 35S:RUBY (Alignment fits perfectly)
-We started working on our reporter systems by sequencing our 35S:RUBY plasmid, creating a glykostock and started to amplificate parts of the plasmid (Paul)
-Isolating P16∆:XVE and 35S:RUBY (Louisa) -Attempt to amplify RUBY in two fragments (Louisa)


05.07. – 11.07.
Isolation and amplification
-Attempting to amplify fragments for tobacco and bacterial cascade (Louisa, Julia, Marie)
-Isolation of pK7WG2 (Lucas)
-Amplification of lexA (Marie)
-Attempting to amplify pK7WG2 in two parts (Louisa)

12.07. – 18.07.
Testing chemicals in bacteria
Preliminary measurement of lethal concentrations on TDG, DIMP, DEMP, MPA (Jacob)
-Testing the lethal concentrations of DEMP, MPA, DIMP, TDG on E.coli DH5a expressing mRFP, through growth measurement in the biolector (Tim, Marvin, Matthes)
-Further attempts to amplify fragments of the signaling cascade (Louisa)

19.07. – 25.07.
-We received agrobacterium and prepared medium Attempting to amplify fragments of signaling cascade and optimise PCR conditions (Louisa)
-Generating new chemocompetent cells with different protocol and testing them with heat shock transformation (Louisa)
-Isolating SUPERR:sXVE (Paul)
-Attempting to join together the amplified RUBY amplificates through GeneSOEing (Julia)


02.08. – 08.08.
Improve a part
- We started our labwork for improve a part by cultivating DH5a containing pRSETb with mCRISPRed and Isolating the plasmid for further cloning steps (Lennart, Tim)
- starting cloning for in vitro tests and improve a part Transformation of Agrobacterium with 35S:RUBY and lexA:RUBY

09.08. – 15.08.
- agroinfiltration of N.benthamiana with 35S:RUBY and are quite happy with the results
- Further cloning efforts for our signaling cascade, improve a part (Tim, Louisa, Lucas, Marie)
- amplification of fragments for Mobius assembly (Paul)

16.08. – 22.08.
Betalain-extraction from Tobacco
- Betalain-extraction from tobacco plants infiltrated with RUBY (Paul, Julia)
- Preparation of second hydroculture experiment (Matthes, Jacob)
- Start of cloning efforts for our library (Lennart)
- Further optimization attempts for amplification of our signaling cascade and cloning efforts (Louisa)

23.08. – 29.08.
First hydroculture
- Addition of our chemicals to the first hydroculture with a longer runtime and sampling and documentation in regular intervals (Jacob, Matthes)
-Gibson assembly of tobacco signaling casacade and screening efforts (Julia, Louisa)

30.08. – 05.09.
Mobius Assembly
-Plasmid isolation and PCRs for mobius assembly (Julia, Marie)
-cloning efforts for improve a part (Tim, Lucas) - screening and inducing clones from bacterial signaling cascade (Tim, Lucas)


06.09. – 12.09.
Expression of mCRISPRed -preparation of second hydroculture and start of freeze drying of the samples from first culture (Matthes, Jacob)
- Amplification, and cloning of RBP, BTCA-R and DIMP-R for receptor expression and library (Lennart, Tim)
-Expression and Purification of mCRISPRed (Lucas)
- Testing lethal BTCA-Concentrations for bacteria with the Biolector (Marvin)

13.09. – 19.09
RNA-iso and GC-MS preparation
- Agroinfiltration of Tobacco with 35S:RUBY, 35S:RUBY and pK7WG2-P4H2-GFP (Julia, Marie, Paul)
- RNA-Isolation, Betalain-extraction and PAM-Measurment of infiltrated Tobacco leaves and addition of estrogen
- Transformation of BL21 DE3 with DIMP-R and BTCA-R (Jonas, Lennart)
- Preparation of GC-MS samples in the lyophylle and cell homogenizer

20.09. – 26.09.
GC/MS is running - Amplification of signaling cascade was optimised with touchdown-PCR (Tim)
- RNA-Isolation of infiltrated plants (Mareike)
- Amplification and Mobius-assembly of reporter constructs (Marie)
- GC/MS was set up and started (Jacob, Matthes)
- Expression and Purification of mRuby3 (Lucas)

27.09. – 03.10.
Concluding improve a part
- Bradford assay for mRuby3 and mCRISPRed and measuring of fluorescence intensity at different pH conditions at the TECAN reader (Lucas)
– cultivating and expression of BTCA-R in BL21 DE3
- Agroinfiltration of tobacco with 35S:RUBY, 35S:RUBY + pk7WG2:P4H4 and negative control (Julia)
- Mobius Assembly of the tobacco signaling cascade (Julia, Paul, Marie)
-Repeating lethal BTCA concentration tests in biolector (Marvin)
- Gibson assembly of bacterial signaling cascade and screening with colony PCR (Tim)
- Induction of positive colonies with Arabinose and Ribose and fluorescence measurement with TECAN (Tim)
- Preparation of infiltrated plant samples for metabolome analysis (Marvin)


04.10. – 10.10.
Measuring fluorescence with CLSM
– Metabolome extraction from infiltrated plant samples and measurement in GC-MS (Marvin)
- RNA-isolation from infiltrated plant samples Bacterial signaling cascade – chemotaxis tests with ribose, measuring fluorescence of different of bacteria under different conditions (Tim)
– Expression of DIMP and purification of DIMP and BTCA under native and deanatured conditions and running SDS-PAGE with samples from purification (Lucas)

11.10. – 17.10.
Testing BTCA-Receptor with SPR
– testing of BTCA-R with surface plasmon resonance spectrometer (Lucas,Tim)
– co-transformation of BL21 with pJOE:RBP & cascade plasmid and induction of liquid cultures and repeating chemoxtaxis experiment (Tim)
– transformation of Agrobacterium with reporter and signaling cascade constructs, hydroculture of tobacco with BTCA, infiltrating hydrocultured plants with Agrobacterium carrying lexA:RUBY, ANTHOS and signaling cascade

18.10. – 21.10.
Characterising the signaling cascade
– inducing co-transformed bacteria in liquid culture and measuring fluorescence with superresolution microscopy (Tim)
– measuring and comparing growth of bacteria with mRuby3 and mCRISPRed (Lucas)