- You may prepare a batch of electrocompetent cells instead of
using the commercially prepared cells. Grow overnight at
37 C a 5 ml LB culture of theE. colicells that you need to
use (for XL1 Blue, grow in LB supplemented with 10μg/ml
tetracycline). Inoculate a 100 ml LB culture the next day with
200 μl of the starter culture. Grow the culture at 37C with
shaking until the OD at 600 nm reaches 0.4–0.8. Harvest the
cells by centrifugation at 6000gat 4C for 10 min. Gently
resuspend the cells in ice-cold water with 10% (vol/vol) glyc-
erol. Do not vortex to resuspend the cells. Repeat this step
three times. Resuspend the cells in 500μl ice-cold water with
10% (vol/vol) glycerol and aliquot 100μl in separate tubes.
Freeze the tubes at 80 C. The cells will be stable for at least
3 years. Thaw cells on ice prior to use. - The mutation frequency of the protocol used for directed
evolution is three mutations per kb. You may increase or
decrease the mutation rate by varying the amount of template.
Please refer to the GeneMorph II Random Mutagenesis Kit
(Agilent) for more information.
References
- Choi KH, Schweizer HP (2006) mini-Tn7 inser-
tion in bacteria with single attTn7 sites: example
Pseudomonas aeruginosa. Nat Protoc 1:153–161 - Holm L, Sander C (1997) An evolutionary trea-
sure: unification of a broad set of amidohydro-
lases related to urease. Proteins 28:72–82 - Pegg SC, Brown SD, Ojha S, Seffernick J, Meng
EC, Morris JH et al (2006) Leveraging enzyme
structure-function relationships for functional
inference and experimental design: the
structure-function linkage database. Biochemis-
try 45:2545–2555 - Seibert CM, Raushel FM (2005) Structural and
catalytic diversity within the amidohydrolase
superfamily. Biochemistry 44:6383–6391 - Afriat L, Roodveldt C, Manco G, Tawfik DS
(2006) The latent promiscuity of newly
identified microbial lactonases is linked to a
recently diverged phosphotriesterase. Biochem-
istry 45:13677–13686- Camilli A, Bassler BL (2006) Bacterial small-
molecule signaling pathways. Science
311:1113–1116 - Dong YH, Wang LH, Xu JL, Zhang HB, Zhang
XF, Zhang LH (2001) Quenching quorum-
sensing-dependent bacterial infection by an
N-acyl homoserine lactonase. Nature
411:813–817 - Guzman LM, Belin D, Carson MJ, Beckwith J
(1995) Tight regulation, modulation, and high-
level expression by vectors containing the arabi-
nose PBAD promoter. J Bacteriol
177:4121–4130
Directed Evolution of Quorum Quenching Enzymes 323