54
1. Wright CM, Wright RC, Eshleman JR,
Ostermeier M (2011) A protein therapeutic
modality founded on molecular regulation. Proc
Natl Acad Sci U S A 108(39):16206–16211
2. Alicea I, Marvin JS, Miklos AE, Ellington AD,
Looger LL, Schreiter ER (2011) Structure of
the Escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors. J Mol Biol 414(3):356–369
3. Deuschle K, Fehr M, Hilpert M, Lager I,
Lalonde S, Looger LL, Okumoto S, Persson J,
Schmidt A, Frommer WB (2005) Genetically
encoded sensors for metabolites. Cytometry A
64(1):3–9
4. Deuschle K, Okumoto S, Fehr M, Looger LL,
Kozhukh L, Frommer WB (2005) Construction
and optimization of a family of genetically
encoded metabolite sensors by semirational protein engineering. Protein Sci 14(9):2304–2314
5. Ribeiro LF, Nicholes N, Tullman J, Ribeiro
LFC, Fuzo CA, Vieira DS, Furtado GP,
Ostermeier M, Ward RJ (2015) Insertion of a
xylanase in xylose binding protein results in a
xylose-stimulated xylanase. Biotechnol Biofuels
8:118
6. Dagliyan O, Shirvanyants D, Karginov AV,
Ding F, Fee L, Chandrasekaran SN, Freisinger
CM, Smolen GA, Huttenlocher A, Hahn KM,
Dokholyan NV (2013) Rational design of a
ligand-controlled protein conformational
switch. Proc Natl Acad Sci U S A 110(17):
6800–6804
7. Guntas G, Mansell TJ, Kim JR, Ostermeier M
(2005) Directed evolution of protein switches
and their application to the creation of ligandbinding proteins. Proc Natl Acad Sci U S A
102(32):11224–11229
8. Tullman J, Guntas G, Dumont M, Ostermeier
M (2011) Protein switches identified from
diverse insertion libraries created using S1
nuclease digestion of supercoiled-form plasmid
DNA. Biotechnol Bioeng 108(11):2535–2543
9. Tullman J, Nicholes N, Dumont MR, Ribeiro
LF, Ostermeier M (2015) Enzymatic protein
switches built from paralogous input domains.
Biotechnol Bioeng 9999:1–7
24. You can use any high-efficiency competent cells that suit your
system. We recommend NEB 5-alpha cells.
25. Check to make sure the cell/DNA sample reaches the bottom
of the cuvette, that it does not have any bubbles and that it is
making contact with both electrodes within the cuvette.
26. Make one large 245 × 245 mm plate (approximately 250 mL
of LB agar) and at least three 10 cm petri dishes of LB agar
(approximately 25 mL of LB agar/plate).
27. If you used multiplex inverse PCR to prepare your vector, you
want the number of transformants to be >5 times the number of
possible variants to have a high probability that the library contains all possible variants (assuming that each library member is
expected to appear at the same frequency). For an in- depth
discussion of this topic, see Bosley and Ostermeier [20].
28. Both DNase I and S1 Nuclease generate libraries with deletions
(and occasionally duplications) distributed along the acceptor
sequence. These deletions contribute to the sequence variability
of the library, potentially generating relevant diversity for the
creation of new properties in the chimeric proteins [11, 19].
Library construction using S1 is usually easier and results in
smaller deletions since S1 nuclease digestion of plasmid DNA is
halted after the first double-stranded break [10]. However, S1
digestion can be heavily biased to occur in inverted repeats
regions in the plasmid, if they are present [8].
References
Lucas F. Ribeiro et al.
1. Wright CM, Wright RC, Eshleman JR,
Ostermeier M (2011) A protein therapeutic
modality founded on molecular regulation. Proc
Natl Acad Sci U S A 108(39):16206–16211
2. Alicea I, Marvin JS, Miklos AE, Ellington AD,
Looger LL, Schreiter ER (2011) Structure of
the Escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors. J Mol Biol 414(3):356–369
3. Deuschle K, Fehr M, Hilpert M, Lager I,
Lalonde S, Looger LL, Okumoto S, Persson J,
Schmidt A, Frommer WB (2005) Genetically
encoded sensors for metabolites. Cytometry A
64(1):3–9
4. Deuschle K, Okumoto S, Fehr M, Looger LL,
Kozhukh L, Frommer WB (2005) Construction
and optimization of a family of genetically
encoded metabolite sensors by semirational protein engineering. Protein Sci 14(9):2304–2314
5. Ribeiro LF, Nicholes N, Tullman J, Ribeiro
LFC, Fuzo CA, Vieira DS, Furtado GP,
Ostermeier M, Ward RJ (2015) Insertion of a
xylanase in xylose binding protein results in a
xylose-stimulated xylanase. Biotechnol Biofuels
8:118
6. Dagliyan O, Shirvanyants D, Karginov AV,
Ding F, Fee L, Chandrasekaran SN, Freisinger
CM, Smolen GA, Huttenlocher A, Hahn KM,
Dokholyan NV (2013) Rational design of a
ligand-controlled protein conformational
switch. Proc Natl Acad Sci U S A 110(17):
6800–6804
7. Guntas G, Mansell TJ, Kim JR, Ostermeier M
(2005) Directed evolution of protein switches
and their application to the creation of ligandbinding proteins. Proc Natl Acad Sci U S A
102(32):11224–11229
8. Tullman J, Guntas G, Dumont M, Ostermeier
M (2011) Protein switches identified from
diverse insertion libraries created using S1
nuclease digestion of supercoiled-form plasmid
DNA. Biotechnol Bioeng 108(11):2535–2543
9. Tullman J, Nicholes N, Dumont MR, Ribeiro
LF, Ostermeier M (2015) Enzymatic protein
switches built from paralogous input domains.
Biotechnol Bioeng 9999:1–7
24. You can use any high-efficiency competent cells that suit your
system. We recommend NEB 5-alpha cells.
25. Check to make sure the cell/DNA sample reaches the bottom
of the cuvette, that it does not have any bubbles and that it is
making contact with both electrodes within the cuvette.
26. Make one large 245 × 245 mm plate (approximately 250 mL
of LB agar) and at least three 10 cm petri dishes of LB agar
(approximately 25 mL of LB agar/plate).
27. If you used multiplex inverse PCR to prepare your vector, you
want the number of transformants to be >5 times the number of
possible variants to have a high probability that the library contains all possible variants (assuming that each library member is
expected to appear at the same frequency). For an in- depth
discussion of this topic, see Bosley and Ostermeier [20].
28. Both DNase I and S1 Nuclease generate libraries with deletions
(and occasionally duplications) distributed along the acceptor
sequence. These deletions contribute to the sequence variability
of the library, potentially generating relevant diversity for the
creation of new properties in the chimeric proteins [11, 19].
Library construction using S1 is usually easier and results in
smaller deletions since S1 nuclease digestion of plasmid DNA is
halted after the first double-stranded break [10]. However, S1
digestion can be heavily biased to occur in inverted repeats
regions in the plasmid, if they are present [8].
References
Lucas F. Ribeiro et al.