Figure 1.
Constructs used for heterologous gene expression from the nuclear genome of C. reinhardtii.
Vector maps represent the constructs used in the study including (A) PAR1::Ble-GFP, (B) PAR4::Ble-2A-GFP, (C) PAR4::Ble-2A-xyn1, (D) pHyg PAR4::Xyn1, and (E) PAR4::Ble-2A-SP-Xyn1. 2A, FMDV 2A peptide, ↑ designates the site of cleavage. Term, rbcs2 3′ terminator. Signal peptide (SP), C.r. ars1 secretion signal sequence.
Figure 2.
FMDV 2A peptide ‘cleavage’ in C. reinhardtii.
A. SDS-PAGE in-gel fluorescence analysis of total protein isolated from transgenic lines expressing ble-GFP or ble-2A-GFP. Labeled bands represent in-gel fluorescence signals of the respective heterologous proteins. B. Microscopy images of GFP signals from transgenic cells expressing ble-GFP or ble-2A-GFP.
Figure 3.
Expression and activity of T. reesei Xyn1 from the ble2A vector in C. reinhardtii.
A. PCR analysis of whole cell lysates from the parental cc1690 (WT) and ble2A-xyn1 strains with primers specific to xyn1 reveals that the transformant is stably transformed. A plasmid containing ble2A-xyn1 is included as a positive control. The expected size of the PCR product is 579 bps. B. Immunoblot analysis of 25 µg of total soluble protein isolated from cc1690 (WT), and a transgenic line transformed with ble2A-xyn1. C. Quantitative immunoblot of monomeric Xyn1. The indicated amount of total soluble protein from the ble2A-xyn strain (right) was compared against a dilution series of purified Xyn1 (left). Densitometric analysis of immunoblot signals using the ImageJ software indicates that Xyn1 accumulates to approximately 0.25% of total soluble protein. The marker (M) shown represents the 26 kDa marker protein. D. Quantitation of xylanase activity in the lysates of C. reinhardtii. Xylanase activity, as measured by the accumulation of the fluorogenic xylanase product DiFMU and represented in relative fluorescence units (RFU), in 50 µg of total soluble protein from the ble2A-xyn1 transgenic line (CrXyn1) is compared to 1 mU, 5 mU, and 10 mU of commercially available Xylanase (A. niger, Megazyme). WT (cc1690) lysate is included as a negative control. Hydrolysis reactions were allowed to proceed for 5 minutes and fluorescence was measured in a Tecan microplate reader (ex360 nm/em460 nm). Data points represent averages of triplicate measurements with error bars representing ± one standard deviation (SD).
Figure 4.
Co-expression of Xyn1 with Ble-2A leads to the selection of transformants with higher xylanase activity.
A. PCR analysis for the presence of xyn1, in 6 independent clones (cl) transformed with PAR4::xyn1 or PAR4::ble2A-xyn1. The expected size of the PCR product is 579 bps. WT, parental strain cc1690; NTC, no template control; P, plasmid positive control; M, 1 kb+ marker. B. Comparison of xylanase activities in 6 independent clones (cl) transformed with xyn1 (left) or ble-2A-xyn1 (right). Xylanase activity is represented as rate of accumulation of the fluorogenic xylanase product DiFMU in µmol/min. Xylanase reactions contain 50 µg of total soluble protein from each clone. Data points represent averages of triplicate measurements with error bars representing ± one SD. Wild type (WT, cc1690) is also included for comparison.
Figure 5.
Insertion of a secretion signal peptide between FMDV 2A and Xyn1 leads to robust accumulation of functional Xyn1 in the culture media.
A. Comparison of intracellular and extracellular xylanase activity for the ble2A-xyn1 (Xyn1) and ble2A-SP-xyn1 (SP-Xyn1) transgenic lines. Intracellular (cells, grey) and extracellular (media, blue) xylanase activities are represented as a percent of total activity for each strain. Both lines were grown in triplicate cultures and the data represents the average percentage of the triplicate cultures. Xylanase reactions were allowed to continue for 15 minutes prior to measuring relative fluorescence. B. Immunoblot analysis of protein prepared from the cells (c) and media (m). Equal volumes were loaded in each well. The size markers represent 36 kDa and 40 kDa (note: the ladder used runs approximately 10 kDa higher on this particular type of SDS-PAGE gel, thus the estimated sizes of cytosolic and secreted xylanase are 26 kDa and 28 kDa, respectively). Wild type (WT, cc3395) is also included for comparison. C and D. Extracellular xylanase activity (D) and cell concentration (C) were monitored over the course of four days. Three biological replicates (a–c) of ble2A-SP-xyn1 and the parental strain, cc3395, were inoculated at 0.8×10∧6 cells per ml and allowed to grow to saturation. Xylanase activity is represented as rate of accumulation of the fluorogenic xylanase product DiFMU in µmol/min/ml of culture media. Xylanase reactions contain 45 µls of culture media. Data points represent averages of triplicate measurements.
Table 1.
Filtering Criteria for autovalidation of database search results.