Download Supplementary Information Surface display of small peptides on

Supplementary Information
Surface display of small peptides on Escherichia coli for
enhanced calcite (CaCO3) precipitation rates
Tushar N Patel1, Ah-Hyung Alissa Park2, and Scott Banta1*
1Department
2Department
of Chemical Engineering, Columbia University, New York, NY 10027
of Earth and Environmental Engineering, Columbia University, New York,
NY 10027
*Corresponding author: 500 W 120th St, New York, NY 10027
Phone: (212) 854-7531; Fax: (212) 854-3054
[email protected]
Table S1 – Oligonucleotide sequences for eCPX cloning
Oligonucleotide
Description
TP49
eCPX Forward Primer
TP50
Reverse Ultramer (FLAG)
(N-DYKDDDDK-C)
TP57
Reverse Ultramer (D20-FLAG)
(N-(D)20-C)
TP58
Reverse Ultramer (GPA-FLAG)
(N-PEVPEGAFDTAI-C)
TP59
Reverse Ultramer (FLK-FLAG)
(N-(FLK)4-C)
Sequence
5’-TCG CAA CTC TCT ACT GTT TCT
CCA TAC CCG-3’
5’-AGT CAG GGC CAC CTT GGC CTT
ATT ATT TGT CAT CAT CGT CTT
TAT AAT CCT GGC CGC TCT GGC
CGC CGC TTG CAG TAC GGC TTT
TCT CGG TGT AA-3’
5’-AGT CAG GGC CAC CTT GGC CTT
ATT ATT TGT CAT CAT CGT CTT
TAT AAT CGC TGC CGC CGC CAT
CGT CAT CGT CAT CAT CGT CAT
CGT CAT CAT CGT CAT CGT CAT
CAT CGT CAT CGT CAT CCT GGC
CGC TCT GGC CGC CGC TTG CAG
TAC GGC TTT TCT CGG TGT AA-3’
5’-AGT CAG GGC CAC CTT GGC CTT
ATT ATT TGT CAT CAT CGT CTT
TAT AAT CGC TGC CGC CGC CAA
TCG CGG TAT CAA ACG CGC CTT
CTG GCA CTT CCG GCT GGC CGC
TCT GGC CGC CGC TTG CAG TAC
GGC TTT TCT CGG TGT AA-3’
5’-AGT CAG GGC CAC CTT GGC CTT
ATT ATT TGT CAT CAT CGT CTT
TAT AAT CGC TGC CGC CGC CTT
TCA GGA ACT TGA GAA ATT TCA
GGA ATT TCA GAA ACT GGC CGC
TCT GGC CGC CGC TTG CAG TAC
GGC TTT TCT CGG TGT AA -3’
TP49 does not contain a recognition site for SfiI. This is because the oligo binds upstream of the
eCPX gene and the 5’ SfiI recognition site in the plasmid, which gets included upon
amplification using TP49. The SfiI recognition sites in TP43, TP57, TP58, and TP59 are
underlined.
Figure S1 – Flow cytometry of cells labeled with anti-FLAG-FITC conjugated monoclonal
antibody. After expression, 0.5 mL of the cell culture was centrifuged at maximum speed
(13000 rpm) for 1 minute. The pellet was then washed 2 times with 1xPBS by resuspending the
cells in the buffer and then pelleting them via centrifugation. The washed pellet was
resuspended in 0.5 mL 1xPBS supplemented with 2.5 µL of the antibody and 5 µL of 100xBSA.
After incubation at room temperature for 20 minutes with gentle agitation, the cells were washed
twice with 1xPBS. The washed pellet was resuspended in 2 mL of 1xPBS and analyzed on a
FACSCanto II Flow Cytometer (BD Biosciences, Franklin Lakes, NJ). (A) BLR, (B) BLRFLAG, (C) BLR-GPA, and (D) BLR-FLK cells labeled with anti-FLAG-FITC antibody.
Figure S2 – Flow cytometry of (A) BLR, (B) BLR-GPA, (C) BLR-pCab/GPA, and (D) BLRpCam/GPA cells labeled with anti-FLAG-FITC antibody using the protocol described in Figure
S1.
Figure S3 – Turbidity (Abs @ 600 nm) against time (solid lines) and Hill fits (dashed lines) for
samples measured in the presence of BLR cells.
Figure S4 – Turbidity (Abs @ 600 nm) against time (solid lines) and Hill fits (dashed lines) for
samples measured in the presence of BLR-GPA cells.
Figure S5 – Turbidity (Abs @ 600 nm) against time (solid lines) and Hill fits (dashed lines) for
samples measured in the presence of BLR-pCab cells.
Figure S6 – Turbidity (Abs @ 600 nm) against time (solid lines) and Hill fits (dashed lines) for
samples measured in the presence of BLR-pCab/GPA cells.
Figure S7 – Turbidity (Abs @ 600 nm) against time (solid lines) and Hill fits (dashed lines) for
samples measured in the presence of BLR-pCam cells.
Figure S8 - Turbidity (Abs @ 600 nm) against time (solid lines) and Hill fits (dashed lines) for
samples measured in the presence of BLR-pCam/GPA cells.