111
7. Dulbecco’s Modified Eagle Medium (DMEM) Glutamax
medium supplemented with 10% fetal bovine serum.
8. Hank’s Balanced Salt Solution (HBSS) supplemented with
10 mg/mL of BSA.
3 Methods
Standard methods of cloning can be used to construct the expression
plasmids for the recombinant fusion proteins. We initially utilized
restriction-enzyme cloning and ligation methods, but have recently
started to use newer ligation methods such as Gibson assembly [26].
We advise using a pET51b(+) (Novagen) expression vector
that contains a double-purification system of a His-tag and a Streptag. Other purification systems can be used, but it is important to
use affinity purification tags on both the N- and C-termini of the
protein to prevent purifying truncated versions of the sensor. We
provide here a general protocol for the expression of sensors, but
modification of the method may be necessary if the binding protein is unstable or insoluble.
1. Clone the sensor into a bacterial expression vector using standard cloning methods (see Note 1).
2. Transform RosettaGami DE3 pLysS E. coli cells for protein
production, plate cells onto selective LB agar medium, and let
colonies grow at 37 °C overnight.
3. Pick a single colony and use it to inoculate 2 mL of selective
LB medium and allow the bacteria to grow overnight, with
shaking at 220 rpm at 37 °C.
4. Inoculate 1 L selective LB medium with 1 mL of the preculture and incubate (220 rpm at 37 °C) until an OD 600nm of 0.60-8 is reached.
5. Cool the incubator to the expression temperature and induce
the expression of the protein depending on the promoter used
(see Note 2).
6. After expression, harvest the cells by centrifugation and resuspend the bacteria in a total of 30 mL of Ni
2+
-NTA lysis buffer
supplemented with 1 mM PMSF and 0.25 mg/mL of
lysozyme.
7. Lyse cells by sonication on ice.
8. Centrifuge at 40,000 × g for 10 min at 4 °C to remove cell
debris and insoluble proteins.
9. Incubate the supernatant with 1 mL Ni
2+
-NTA agarose slurry
under agitation for 1 h at 4 °C. Then centrifuge gently and
transfer the resin onto a propylene column.
3.1 Generation
of Fusion Proteins
for Expression in E.
coli
SNIFITS and LUCIDs: Semi-Synthetic Modular Biosensors
7. Dulbecco’s Modified Eagle Medium (DMEM) Glutamax
medium supplemented with 10% fetal bovine serum.
8. Hank’s Balanced Salt Solution (HBSS) supplemented with
10 mg/mL of BSA.
3 Methods
Standard methods of cloning can be used to construct the expression
plasmids for the recombinant fusion proteins. We initially utilized
restriction-enzyme cloning and ligation methods, but have recently
started to use newer ligation methods such as Gibson assembly [26].
We advise using a pET51b(+) (Novagen) expression vector
that contains a double-purification system of a His-tag and a Streptag. Other purification systems can be used, but it is important to
use affinity purification tags on both the N- and C-termini of the
protein to prevent purifying truncated versions of the sensor. We
provide here a general protocol for the expression of sensors, but
modification of the method may be necessary if the binding protein is unstable or insoluble.
1. Clone the sensor into a bacterial expression vector using standard cloning methods (see Note 1).
2. Transform RosettaGami DE3 pLysS E. coli cells for protein
production, plate cells onto selective LB agar medium, and let
colonies grow at 37 °C overnight.
3. Pick a single colony and use it to inoculate 2 mL of selective
LB medium and allow the bacteria to grow overnight, with
shaking at 220 rpm at 37 °C.
4. Inoculate 1 L selective LB medium with 1 mL of the preculture and incubate (220 rpm at 37 °C) until an OD 600nm of 0.60-8 is reached.
5. Cool the incubator to the expression temperature and induce
the expression of the protein depending on the promoter used
(see Note 2).
6. After expression, harvest the cells by centrifugation and resuspend the bacteria in a total of 30 mL of Ni
2+
-NTA lysis buffer
supplemented with 1 mM PMSF and 0.25 mg/mL of
lysozyme.
7. Lyse cells by sonication on ice.
8. Centrifuge at 40,000 × g for 10 min at 4 °C to remove cell
debris and insoluble proteins.
9. Incubate the supernatant with 1 mL Ni
2+
-NTA agarose slurry
under agitation for 1 h at 4 °C. Then centrifuge gently and
transfer the resin onto a propylene column.
3.1 Generation
of Fusion Proteins
for Expression in E.
coli
SNIFITS and LUCIDs: Semi-Synthetic Modular Biosensors