La comunidad académica de los filósofos colombianos.

A M. ruminantium M1 phage display library was constructed in the phage display vector pYW01, which enables recombinant proteins to be produced as fusions to the C domain of phage pIII protein (Gagic et al., 2013) (Figure 3.1A). The primary library contained 3.2×108 independent clones containing insert sizes ranging from 1 to 4 kb. Theoretically, complete coverage of the M1 genome (2.93 Mb) (Leahy et al., 2010) requires a minimum of 6.7×103 clones containing an average insert length of 2 kb (Jacobsson et al., 2003) (Equation 3.1)]; therefore, it is estimated that the genome coverage of this library is 4.8×104. This library was infected with helper phage VCSM13 to generate a master library of PPs, displaying the M1 proteome. The master library was used for affinity screening to identify adhesins that bind protozoa.

Equation 3.1. Minimum number of clones required for coverage of a given genome of size b, from Jacobsson et al. (2003).

N denotes the number of clones required, P denotes the probability that a given fragment is

present in the library, a denotes the average fragment size, and b denotes the genome size. To

estimate the number of clones required to represent the M1 genome, P = 0.99, a = 2000,

b = 2,930,000)

= ln (1 − )

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Figure 3.1. Plasmid map of phagemid vector pYW01 and pMru_1499A.

Panel A shows the phagemid vector pYW01 used to create the M1 phage display library. A chloramphenicol (Cm) resistance cassette, phage origin of replication, and plasmid origin of

replication (shaded in green) can be found in the phagemid. Inserts were cloned into the SmaI

restriction site. The gene sequences encoding a c-myc tag and the C domain of phage protein pIII are downstream of the restriction site, which enables recombinant fusion proteins to be

generated. Panel B shows the phagemid vector pMru_1499A _{which encodes the affinity-selected}

protein Mru_1499A_{. The insert sequence encodes an endogenous signal sequence. Also, the insert}

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As rumen protozoa are fragile, sensitive to oxygen, and difficult to maintain in culture, cells harvested from the sheep rumen contents were formalin-fixed prior to affinity screening to prevent cell lysis and to preserve the integrity of their cell surfaces. It is known that formalin fixation of live cells causes cross-linking of cell-surface proteins and can reduce the diversity of binders recovered (Qiao et al., 2012); however this step was necessary to prevent disintegration of cells. This preparation was used as “bait” or ligand to bind PPs expressing protein domains to protozoa. During affinity screening, PPs were incubated with protozoa, followed by a series of washes to remove non- specific or background binders. The protozoa-attached PPs were subsequently eluted from bait, collected, and then amplified through infection of the E. coli host.

Plasmid profiles of the library pools corresponding to protozoa-attached PPs were monitored by agarose gel electrophoresis after each round of affinity screening. Before affinity screening, a smear was observed upon agarose gel electrophoresis of purified plasmids due to the random size distribution of inserts in the phage display library. After two rounds of affinity screening, four discrete plasmid bands were observed, indicating enrichment for plasmids of specific sizes (Figure 3.2). The plasmid bands were excised and purified, and then transformed into E. coli host strain TG1. After sequence analysis of 13 clones, a recombinant clone encoding the partial gene sequence of

mru_1499 in frame with the phagemid vector c-myc tag and gIII was selected for further analysis (Figure 3.1B). This clone originated from plasmid band 2 in Figure 3.2. The remaining 12 clones were not pursued as the encoded protein was not in frame with the gene encoding phage protein pIII. The gene mru_1499 was also up-regulated when M1 was co-cultured with rumen bacterium Butyrivibrio proteoclasticus B316 (Leahy et al., 2010). Before the start of this project, the export of an archaeal protein harbouring an archaeal signal sequence was tested in E. coli to confirm that archaeal signal sequences

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are recognized in the bacterial host (data not shown). Here, Mru_1499A contains an archaeal-derived signal sequence, which also shows that the E. coli host can recognize some archaeal signal sequences. Other recombinant clones were “background” clones that contained DNA inserts encoding peptides that were out of the frame with pIII, and therefore not expected to display functional fusion peptides on the surface of the virion. Background clones are frequently observed in affinity-selected phage display libraries (Vodnik et al., 2011). The protein encoded by the 5´ moiety of gene sequence mru_1499

that corresponded to the insert in the recombinant phagemid was designated as Mru_1499A (the ‘A’ stands for affinity selected portion of the gene), and the phagemid vector encoding Mru_1499A is denoted pMru_1499A.

Figure 3.2. Plasmid profile of M1 library before and after panning against protozoal bait.

Lane 1 contains pYW01 plasmid DNA (no insert). Lane 2 contains plasmid DNA extracted from

the primary library. Lanes 3 and 4 contain plasmid DNA extracted from E. coli cells after the first

and second round of panning against protozoal bait, respectively. After two rounds of biopanning, enriched plasmid DNA bands indicated by arrows (Band 1 and Band 2) were

extracted and transformed into E. coli for further analysis. The lower molecular weight bands

were not further pursued, as they likely correspond to empty vector or short inserts that have been enriched due to growth advantages.

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