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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. Colicin activity: OD

10. Colicin activity: GFP

11. 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

12. 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

13. Sender-Part: Results of activity-tests

14. Sender-Part: Negative controll of activity-tests