Download SUPPORTING INFORMATION, Plucinak et al. Figure S1: Protein blot

SUPPORTING INFORMATION, Plucinak et al.
Figure S1: Protein blot analysis of the efficiency of TaV 2A processing in
Chlamydomonas cells. Proteins from cells transformed with mCherry Bler-TaV
2A-mCherry-CIA5 Ep gene construct detected on a protein blot using polyclonal
antibodies raised against a synthetic CIA5 epitope (CIA5 Ep). Lane1) A zeocin
resistant (Bler) culture not expressing mCherry. Lanes 2 and 3) Zeocin resistant
cultures expressing and processing Bler-TaV 2A-mCherry-CIA5 Ep. Expected
sizes of indicated bands are as follows: CIA5 = ~100 kDa, full length
unprocessed Bler- TaV 2A-mCherry-CIA5 Ep = 48.6 kDa, mCherry-CIA5 Ep =
30.4 kDa.
Figure S2: FKB12 complementation assay. A) Zeocin resistant colonies of
originally rapamycin-resistant Chlamydomonas fkb12 mutants transformed with
plasmid pMA4 containing a nonfunctional FMDV 2A coding sequence separating
a zeocin resistance gene and a FKB12 gene (left column) or a separate set of
colonies generated from cells transformed with plasmid pLZ1 containing a
functional FMDV 2A coding sequence separating a zeocin resistance gene and a
FKB12 gene (right column). Colonies were randomly picked to TAP liquid
medium in microtiter plates, incubated overnight, and replica plated onto TAP
plates (top row) and TAP plates containing rapamycin (bottom row). Successful
complementation of fkb12 mutants with a functional FKB12 gene results in loss
of rapamycin resistance. B) A second fkb12 mutant containing a 21 bp deletion
and a 4 bp insertion in the FKB12 gene (bottom DNA sequence) was
transformed with pLZ1 and a set of the resultant zeocin resistant colonies of the
originally rapamycin-resistant Chlamydomonas mutants were randomly picked to
TAP liquid medium in microtiter plates, incubated overnight, and replica plated
onto TAP plates (left) and TAP plates containing rapamycin (right). WT, wild
type.
Figure 2S. See legend next page.
Figure S3: Enlargement of portions of Figure 6A to better visualize
mCherry-tagged Rubisco in the Chloroplast of cells transformed with
plasmid QN4. A) Portions of original image of cells maintained in high CO2
conditions (lower left panel) as contrasted to the exclusive localization of
mCherry-tagged Rubisco in the pyrenoid of the same cells maintained in low CO 2
concentrations (upper left panel). B) Red color enhancement with Photoshop of
images shown in A to more easily visualize the exclusive localization of mCherrytagged Rubisco fluorescence to the pyrenoid (upper left panel) and the patterns
of mCherry-Rubisco redistribution to the chloroplast stroma following shift of cells
from low CO2 to high CO2 conditions (lower left panel).
Figure S4: Evaluation of various selectable markers for compatibility with
the extended FMDV 2A peptide. A) Constructs for testing effects of opposite
orientations of a gene of interest (GOI) and selectable markers in 2A-containing
dicistronic genes. Gluc, Gaussia Luciferase; 2A, extended FMDV 2A peptide;
PsaD Pro, promoter from Chlamydomonas PsaD gene. B) Performance of each
construct in regard to effect on transformation rates, maintenance or loss of
linkage between the GOI (Gluc) and the selectable marker gene, and the
approximate relative luminescence units (RLUs) measured in cultures of at least
10 randomly chosen transformants. Linkage was defined as detection of Gluc
signal in at least 70% of transformants tested. Bler (Zeocinr); Aph8
(Paromomycinr); Aph7 (Hygromycinr); AadA (Spectinomycinr); Arg7
(argininosuccinate lyase; complementation of arginine auxotrophy in arg7
mutants); RbcS2 (Rubisco small subunit 2, complementation of photoautotrophy
in rbcs null mutants); pSP124, standard laboratory construct containing a Bler
gene driven by the RbcS2 gene promoter (used as a control).