Colicins

1
Colicins

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

2
Parts Sender

• Sender Part (BBa_F1610) cloned in pBAD plasmid
• Functional sender -gt no BioBrick standard

luxI C0061
pBAD
B0034
B0010
B0012
3
Parts Sender

• Further plans readjust part to BioBrick standard
• Combine part BBa_F1610 and BBa_I0500 (pBAD
  promotor)
• Possibly clone CFP cassette behind this new
  sender part

luxI C0061
I0500
B0034
B0010
B0012
P(tet)R R0040
luxI C0061
RBS
ECFP
B0010
B0012
I0500
B0034
B0010
B0012
4
Parts Receiver
Source Receiver Part (BBa_T9002)
P(tetR) R0040
luxR C0062
Lux pR R0062
B0034
B0010
B0012
RBS
GFP
B0010
B0012

• Combination of existing receiver part with
  colicin in pSB1A3
• Receiver Part without GFP

P(tetR) R0040
luxR C0062
Lux pR R0062
B0034
B0010
B0012
RBS
BamHI
5
Parts Receiver

• Combine Receiver part without GFP with colicin
  cassettes
• Colicin E1
• Colicin E9

Rec
Col
EcoRI
P(tetR) R0040
luxR C0062
Lux pR R0062
B0034
B0010
B0012
RBS
Bam HI
Colicin E1
T1
kil
Pim
P(tetR) R0040
luxR C0062
Lux pR R0062
atypical lysis protein
Colicin E9
RBS
B0034
B0010
B0012
RBS
Bam HI
T1
E9 imm
6
Parts Colicin

• Primers are available to clone colicin parts into
  a standard BioBrick vector
• Colicin E1
• Kil E1 lysis protein
• E1 imm immunity protein

Colicin E1
kil
E1 imm
7
Parts His-tagged colicins

• Clone of colicin genes in His-tag Vector pQE-30
• Purification of colicin protein
• Quantification of colicin E1 E9 activity
• No BioBrick standard vector
• Commercial vector (Qiagen)

8
What have we done so far further plans

• Cloning of Sender part
• Finished cloning of sender in pBAD
• Next step cloning of F1610 (sender) behind I0500
  (pBAD) -gt ready for registry
• Cloning of Receiver part
• Troubles with first cloning part -gt new cloning
  attempt
• Last step combination of working receiver part
  with a fluorescence gene cassette
• Colicin activity tests on bacteria and eukaryotic
  cells

9
Colicin activity OD
10
Colicin activity GFP