Colicins

1
Colicins

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

2
Outline

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

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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

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pQE-30 Colicin Cloning Current state
problems

• Current state
• 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

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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

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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

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Receiver-Part Strategy current state

• Colicin E9 short Cloning finished,
  standardization and characterization
• Colicin E9 long Cloning finished,
  standardization and characterization

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Receiver-Part Standardization Problems

• Finished receiver part contains false BioBrick
  Prefix and Suffix

Correction in progress
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Colicin activity OD
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Colicin activity GFP
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Sender-Part Strategy

• Working sender part (BBa_F1610) cloned in pBAD
  plasmid -gt 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
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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
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Sender-Part Results of activity-tests
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Sender-Part Negative controll of activity-tests