Colicins Yara Reis Erik Sommer Pascal Krämer Andreas Kühne

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Presentation transcript:

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

Outline Protein purification with pQE-30-colicin pSB1A3-receiver-colicin-cloning Colicin E1 Colicin E9 AI-1 sender pBAD sender Constitutive sender 25.02.2019

pQE-30 – Colicin Cloning: Strategy Clone of colicin genes in His-tag Vector pQE-30 Purification of colicin proteins Quantification of colicin E1 & E9 activity: production rate of our colicin-receiver parameters for modeling group activity on eukaryotic cells 25.02.2019

pQE-30 – Colicin Cloning: Current state & problems 4th cloning attempt, the last three did not work Problems High religation rate of the vector Possibly the lac promoter is to leaky colicin is produced while promoter is not induced the cells kill themselves because they do not have an immunity gene 25.02.2019

pQE-30 – Colicin Cloning: Further plans New cloning attempt and transformation into… … BL-21 cells: Expression strain which contains helper plasmid with stronger supression of lac promoter. … BtuB KO strains … Cotransformation with immunity gen 25.02.2019

Receiver-Part: Strategy & current state Source: GFP-receiver part (BBa_T9002) Combination of existing receiver part with colicin gene cassettes in pSB1A3 Colicin E1: Cloning in progress P(tetR) R0040 B0034 luxR C0062 B0010 B0012 RBS Lux pR R0062 GFP EcoRI + 3 x PstI P(tetR) R0040 B0034 luxR C0062 B0010 B0012 RBS Lux pR R0062 Bam HI Colicin E1 T1 kil 25.02.2019

Receiver-Part: Strategy & current state Colicin E9 short: Cloning finished, standardization and characterization Colicin E9 long: Cloning finished, standardization and characterization P(tetR) R0040 B0034 luxR C0062 B0010 B0012 RBS Lux pR R0062 Bam HI Colicin E9 T1 E9 imm Pim atypical lysis protein EcoRI P(tetR) R0040 B0034 luxR C0062 B0010 B0012 RBS Lux pR R0062 Bam HI Colicin E9 T1 E9 imm Pim atypical lysis protein E5 imm canonical lysis protein Colicin E9 short can be submitted to the registry 25.02.2019

Receiver-Part: Standardization & Problems Finished receiver part contains false BioBrick Prefix and Suffix gaattcgcggccgcttctagatccctatc... cttaagcgccggcgaagatctagggatag... How our prefix looks like… How our suffix looks like… ...aaaacctagactagtagcggccgctgcag ...ttttggatctgatcatcgccggcgacgtc Prefix Receiver… …Colicin Suffix EcoRI NotI XbaI NotI SpeI PstI gaattcgcggccgcttctagaGtccctatc... cttaagcgccggcgaagatctCagggatag... …and how it should look like. …and how it should look like. ...aaaacctagTactagtagcggccgctgcag ...ttttggatcAtgatcatcgccggcgacgtc Prefix Receiver… …Colicin Suffix EcoRI NotI XbaI NotI SpeI PstI Correction in progress 25.02.2019

Colicin activity: OD 25.02.2019

Colicin activity: GFP 25.02.2019

Sender-Part: Strategy Working sender part (BBa_F1610) cloned in pBAD plasmid -> No BioBrick standard Standardization: Combination of BBa_F1610 with standard promoters of the registry pBAD promoter (BBa_I0500) medium constitutive promoter (BBa_J23107) luxI C0061 pBAD B0034 B0010 B0012 pBAD I0500 B0034 luxI C0061 B0010 B0012 Prom J23107 B0034 luxI C0061 B0010 B0012 25.02.2019

Sender-Part: Current state & further plans pBAD sender: Evaluation of cloning With positive clones: Activity test with GFP-receiver (BBa_T9002) for characterization Constitutive sender: Positive clones Activity tests with GFP-receiver are in progress (BBa_T9002) Two improved parts 25.02.2019

Sender-Part: Results of activity-tests 25.02.2019

Sender-Part: Negative controll of activity-tests 25.02.2019