Colicins Yara Reis Erik Sommer Pascal Krmer Andreas

Colicins Yara Reis Erik Sommer Pascal Krämer Andreas Kühne Kathrin Nußbaum Marika Ziesack

Overview • Introduction • Vorstellen der Parts – Sender – Amplifier – Receiver (1 -3) • Colicine (vorhandene) – E 1 – E 9 • AI-1 Induktion • Sender/Amplifier Induktion • Pläne – Klonieren Colicine – Visualisierung – Evtl. eukaryotische Zelllinien (Tumor) 9/29/2020 Killing by Colicin 2

Introduction • Two E. coli Strains – Sender – Killer • Sender – Lux. I expression – AI-1 transfer into the medium • Killer – AI-1 activates Lux p. R (Promotor) Colicin expression 9/29/2020 Killing by Colicin 3

Parts: Sender/Amplifier • Sender Part (BBa_F 1610) • No Promoter • Amplifier Part (BBa_I 15030) 9/29/2020 Killing by Colicin • Positive feedbackloop • Amplified production of AI-1 4

Parts: Receiver • Receiver Part (BBa_T 9002) • • • Promoter is constitutively ON Repression by Tet. R repression inhibited by tetracycline • Receiver Part (BBa_F 2621) • Receiver Part (BBa_F 2622) • • Weak constitutive expression Expression down-regulated by Lux. R • • Repression by Lac. I Induction by IPTG Two molecules of Lux. R protein form a complex with two molecules the signalling compound homoserine lactone (HSL) This complex binds to a palindromic site on the promoter, increasing the rate of transcription from Pr 9/29/2020 Killing by Colicin 5

Colicins • E 9/Im 9 – By University of York (……………) – Plasmids – DNAse • E 1 – Commercial by DSMZ – Cells JC 411 E. coli – Pore-forming • Waiting for some other answers 9/29/2020 Killing by Colicin 6

What have we done so far? • Transformation, Glycerol-Stocks, Mini. Preps – Sender (F 1610) – Amplifier (I 15030) – Receiver (T 9002, F 2621, F 2622) • Check of the receiver part (T 9002) by restriction digest • AI-1 Induction of receiver-cells – Different concentration – Measurement during several hours • Receiver-cell-induction by amplifier-cells – Different ratios of amplifier/receiver – Measurement during several hours 9/29/2020 Killing by Colicin 7

AI-1 Induction of receiver-cells • Different concentrations of AI-1: – 0 M, 1 n. M, 10 n. M, 500 n. M, 50 µM 9/29/2020 Killing by Colicin 8

Receiver-cell-induction by amplifier-cells • Ratio amplifier: receiver – 1: 5, 1: 2, 1: 1, 2: 1, 5: 1 – Receiver concentration constant • • 9/29/2020 Killing by Colicin Maximal induction from 3 to 5 h Low amplifier concentration lead to high induction of GFP in receiver-cells 9

Receiver-cell-induction by amplifier-cells 9/29/2020 Killing by Colicin 10

What will we do in future? • Control of sequences of – Sender (F 1610) – Amplifier (I 15030) – Receiver (F 2621, F 2622) • Cloning of sender-part behind constitutive promoters of different activities (? ) • Cloning of the Colicin behind the receiver-parts 9/29/2020 Killing by Colicin 11

Future Prospects in General • Swarm plates and visualization of sensing and killing • Effect of colicins on eukaryotic cell-lines 9/29/2020 Killing by Colicin 12