Algorithm environment

2.7.1 Construction of pAG2-A!42

The oligonucleotides listed in Table 2.8 were used during vector construction as outlined in this section; BioBrick part sequences (BBa; Canton et al. [2008]) are available at the Registry of Standard Biological Parts (accessed at http://partsregistry.org/).

Table 2.8: Primers used to construct pAG2-A!42.

2.7.1.1 Insertion of mCherry operon

Primers pAG-Via1 and pAG-Via2 were used to PCR amplify (Phusion polymerase, touchdown 61ºC to 52ºC, then annealing at 65ºC) the mCherry operon (BBa_J06702, partnered with the medium-strength promoter BBa_J23101) from pSB3K3-mCherryGFP1plus3. This 958 bp product was inserted into pBADm near its BsaAI site by CPEC Viaduct (see Section 2.2.6) so that the mCherry coding region lay between the amp^R operon and the origin of replication, resulting in pAG-A.

2.7.1.2 Construction and insertion of Library operon

The Library site consisted of the strong promoter BBa_J23119, ribosome binding site BBa_B0034, a start codon straddled by SpeI and AfeI sites, stop codons for all three reading frames, and the rrnB T1/T2 terminator region (for more detail see Appendix 8). Primers pAG-Via3 and pAG-Via4, which overlapped, were extended by PCR amplification (Phusion polymerase, touchdown 62ºC to 53ºC, then annealing at 72ºC), giving a 78 bp product (named A). Primers pAG-Via6 and pAG-Via7 were used to PCR amplify (as above) the rrnB T1/T2 terminator region

from pBADmC (see Section 2.6.7.2), giving a 249 bp product (named B). Product B was re-amplified (as above) using Primers pAG-Via19 and pAG-Via 7, giving a 279 bp product (named C). Products A and C, which overlapped, were stitched together by PCR amplification (as above) using primers pAG-Via3 and pAG-Via7, giving the full-length Library site. This 335 bp product was inserted into pAG-A by CPEC Viaduct (see Section 2.2.6; annealing at 62.5ºC) between the end of the araBAD site’s transcriptional terminator and the start of the amp^R operon, resulting in pAG2-B.

2.7.1.3 Insertion of A!42-EGFP into the araBAD site

Primers pAG-Via8 and pAG-Via9 were used to PCR amplify (Phusion polymerase, touchdown 62ºC to 53ºC, then annealing at 72ºC) A!42-EGFP from pA!42-EGFP.

This 929 bp product was inserted into pAG2-B by CPEC Viaduct (see Section 2.2.6;

annealing at 60ºC) between the araBAD site’s start codon and the PmeI site, resulting in pAG2-A!42. The linker between the A!42 and EGFP domains was GSAGSAAGSGESHMV (15 aa; as per Baine et al., [2009]). See Section 5.2.1.1 for a schematic of pAG2-A!42; the full annotated sequence is shown in Appendix 7.

2.7.2 Construction of pAG2-A!42 controls

The oligonucleotides listed in Table 2.9 were used during vector construction as outlined in this section.

Table 2.9: Primers used to construct pAG2-A!42 controls.

2.7.2.1 Construction of pBADm-EGFP

As a single (green) positive control, EGFP was inserted into the araBAD site of pBADm (see Section 2.6.1.1). Primers EGFP-Via1 and EGFP-Via2 were used to PCR amplify (Phusion polymerase, touchdown 65ºC to 56ºC, then annealing at 72ºC) EGFP from pET28-A7-EGFP. This 744 bp product was inserted into pBADm by CPEC Viaduct (see Section 2.2.6; annealing at 65ºC) between the NcoI and XbaI sites of the araBAD region, resulting in pBADm-EGFP. The sequence ran on into the pBADm C-terminal hexa-histidine tag (17 aa extra).

2.7.2.2 Insertion of EGFP into Library site of pAG2-A!42

To test the activity of the Library site, EGFP was inserted as a reporter. Primers pAG-Via21 and Cherry-REV2 were used to PCR amplify (Phusion polymerase, touchdown 60ºC to 51ºC, then annealing at 58ºC) EGFP from pET28-A7-EGFP (the same sequence as Baine et al. [2009]), incorporating a 5’ SpeI and 3’ XbaI site.

This 757 bp product was subsequently digested with SpeI/XbaI (which share compatible sticky ends) and ligated into dephosphorylated pAG2-B via the SpeI Library site, resulting in pAG2-B/EGFP.

2.7.2.3 Construction of pAG2-GM6

pAG2-A!42 was modified to incorporate the non-aggregating A!42 mutant GM6 (Wurth et al., 2002) as a positive control. Primers pAG-Via8 and pAG-Via10 were used to PCR amplify (Phusion polymerase, touchdown 62ºC to 53ºC, then annealing at 72ºC) GM6 from pSALect-GM6. This 151 bp product was inserted into pAG2-A!42 by CPEC Viaduct (see Section 2.2.6; annealing at 51ºC) to replace the A!42 domain, resulting in pAG2-GM6.

2.7.2.4 Insertion of Peptide 2 (Pep2) and AavLEA1 into pAG2-A!42

The coding sequences for Pep2 (Baine et al., 2009) and AavLEA1 (Browne et al., 2002) were inserted into the Library site of pAG2-A!42 to use as antiaggregant controls. To create the Pep2 coding sequence, complimentary oligonucleotides p2F and p2R were annealed by heating 20 !L of a 25 !M solution to 95ºC for 5 min and subsequently cooling from 80ºC to 60ºC at 0.5ºC/min. This 50 bp product was PCR amplified (Phusion polymerase, touchdown 62ºC to 53ºC, then annealing at 72ºC)

using primers pAG-Via23 and pAG-Via16. The resulting 92 bp product was inserted into pAG2-A!42 by CPEC Viaduct (see Section 2.2.6; annealing at 51ºC) between the Library site’s start codon and first stop codon, resulting in pAG2-A!42-Pep2.

Primers pAG-Via25 and pAG-Via18 were used to PCR amplify (as above) AavLEA1 from pET15-AavLEA1. This 476 bp product was inserted into pAG2-A!42 as per Pep2 above, resulting in pAG2-A!42-AavLEA1.

2.7.3 Insertion of random DNA library into pAG2-A!42

Oligonucleotides (see Table 2.10) coding for a maximum of either 12 or 24 random amino acids (13 or 25 including the initiating methionine) were PCR amplified (from 10 pmol of template; Phusion polymerase, annealing at 57ºC) using primers pAGIns-FWD and pAGIns-REV. Only 15 amplification cycles were used in order to minimise any loss of product diversity. These 74 and 110 bp products were subsequently digested with SpeI/AfeI and ligated into 200 ng of dephosphorylated, gel extracted pAG2-A!42 via the same sites at a vector:insert molar ratio of 1:5 using T4 DNA ligase (Fermentas GmbH) at 23ºC for 1 hour (40 !L total volume).

Ligase was inactivated prior to transformation via storage of ligation mixture at -20ºC. The resulting libraries were named pAG2-A!42-74 (13 aa peptide) and pAG2-A!42-110 (25 aa peptide). For each desired agar plate, 1.25 !L of ligation mixture was transformed into ~12.5 !L of chemically-competent E. coli DH10B.

Each transformation was plated on LB agar containing 0.05% arabinose.

Table 2.10: Oligonucleotides used to create random DNA libraries. N, any base pair.

2.7.4 Microscopy

Bright field and fluorescence microscopy were undertaken on an Optiphot-2 microscope with an EFD-3 epifluorescence system (Nikon Corp., Tokyo, Japan).

Images were captured using a DS-2Mv camera system (Nikon Corp.). EGFP had peak absorption and emission wavelengths of 488 nm and 509 nm, while mCherry was 587 nm and 610 nm respectively (Takara Bio Inc.). For EGFP, a B-2A filter block was used (excitation 470 +/- 20 nm, emission >520 nm); for mCherry, G-2A (excitation 535 +/- 25 nm, emission >590 nm). Bleed-through of mCherry fluorescence was observed when using the B-2A filter block for long exposure times; such excitation catches the extreme left-hand shoulder of mCherry’s absorption spectrum, and its full emission spectrum is able to transit the longpass emission filter. No bleed-through of EGFP fluorescence was observed using the G-2A filter block.

2.7.5 Flow cytometry

2.7.5.1 Flow cytometry analysis of E. coli fluorescence

Cultures (3 mL) containing the various induced (see Section 2.3.1) constructs were diluted to approximately 2x10⁷ cfu/mL in phosphate buffered saline (PBS; 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) and analysed using a DxP 8 FACScan II flow cytometer (Cytek Development Inc., Fremont, USA) as per the manufacturer’s instructions. Any cultures exhibiting cell clumping were thoroughly vortexed immediately prior to analysis to aid dispersion. Cells were initially gated on a dot plot by log Forward Scatter (FS) versus log Side Scatter (SS) to select for a homogenous cell population (G1), and G1 subsequently gated by log FS area versus log FS width to select for single cells only, i.e. doublet discrimination (G2). Cells selected by G1 and G2 were individually interrogated for EGFP or mCherry fluorescence using the appropriate single-positive control strains, and a compensation matrix applied to correct for any spectral overlap observed between the two (minimal). For EGFP, excitation at 488 nm was used, with emission monitored at 530 +/- 15 nm; for mCherry, excitation at 561 nm was used, with emission monitored at 615 +/- 12.5 nm. A total of 100,000 events were recorded per sample. Data was analysed using FlowJo v7.6.4 (Tree Star Inc., Ashland, USA).

2.7.5.2 Preparation of libraries for fluorescence-activated cell sorting

Approximately 25,000 colonies (30ºC incubation for ~16 h after transformation [as per Section 2.2.8]) from each of the pAG2-A!42-74 and pAG2-A!42-110 libraries

subsequently normalised by dilution to OD600 = 2.5 (Section 2.3.1). The same was performed for approximately 25,000 colonies containing pAG2-A!42, which was then split into three aliquots and spiked separately with 3 colonies of pAG2-A!42-Pep2, pAG2-A!42-AavLEA1 or pAG2-GM6. These five resuspensions were used as inoculums for induction of expression as per Section 2.3.1.

2.7.5.3 Fluorescence-activated cell sorting (FACS)

Cultures (3 mL) to be analysed were pelleted and resuspended in PBS prior to being diluted further as per Section 2.7.5.1, and sorted on a DakoCytomation MoFlo MLS cell sorter (Beckmann Coulter Inc., Brea, USA) as per the manufacturer’s instructions under the supervision of Mr Nigel Miller (Flow Cytometry Facility, Department of Pathology, University of Cambridge). Cell population gating was as per Section 2.7.5.1, with spectral compensation performed manually by adjusting photomultiplier tube (PMT) gain. For EGFP, excitation at 488 nm was used, with emission monitored at 530 +/- 15 nm; for mCherry, excitation at 568 nm was used, with emission monitored at 613 +/- 10 nm. At least 20,000 total events were recorded per sample. Data was analysed using Summit v4.3.02 (Beckmann Coulter Inc., Brea, USA). For cell sorting, approximately 1x10⁴ positive events (indicated) from 1x10⁶ cells were selected and pooled into 3 mL fresh LB medium. For further enrichment, these pooled cultures were directly grown and induced as per Section 2.3.1 before undergoing additional rounds of FACS (indicated). After the final round, cells were plated out on LB agar.

2.7.5.4 Calculations

To give a measure of the spread of data obtained during flow cytometry, the coefficient of variation (CV) was used (Huber, 2005). CV represents a normalised standard deviation, i.e. the standard deviation divided by the mean, and is commonly converted to a percentage. A more preferred statistic is the robust CV (rCV), which is not as skewed as CV by outlying data, and was calculated using Equation 2.1 using the flow cytometer manufacturer’s software (Section 2.7.5.1).

!

%rCV =100 *1

2

(

)

Equation 2.1: %rCV, robust coefficient of variation (as a percentage).

However, the rCV calculation was not available for the MoFlo MLS cell sorter (Section 2.7.5.3), so CV was used instead.

2.7.6 Identification of pAG2-A!42 library hits on solid medium

EGFP fluorescence from induced colonies was photographed (3 s exposure) using an ImageQuant LAS 4000 system (GE Healthcare UK Ltd.). Excitation was at

~460 nm (blue epifluorescence), emission at 510 nm (510DF10 filter). Images were imported into ImageJ v1.41o (National Institutes of Health, Bethesda, USA), and the

“threshold” function applied to highlight the colonies with the brightest fluorescence.

Any colony hits identified were manually verified by microscopy for both EGFP and mCherry fluorescence.

2.7.7 Bioinformatic analysis of pAG2-A!42 library hits

Peptide sequences were interrogated for pairwise alignment to the A!42 sequence using the European Molecular Biology Open Software Suite (EMBOSS) Matcher v2.0u4 (Rice et al. [2000]; default options used, with 3 alternative matches shown;

accessed at http://www.ebi.ac.uk/Tools/psa/emboss_matcher/), or for multiple sequence alignment to each other using ClustalW2 v2.1 (Larkin et al. [2007]; default options used; accessed at http://www.ebi.ac.uk/Tools/msa/clustalw2/). Open Reading Frame Finder (NCBI; bacterial genetic code used; accessed at http://www.ncbi.nlm.nih.gov/projects/gorf/) was used to search for alternate start codons in insert sequences with short open reading frames originating from the Library site start codon. To predict the percentage of sequences from a random DNA insert library that should incorporate an internal stop codon, Equation 2.2 was utilised (Walker et al., 2001).

% peptides with an internal stop codon=1" 61 encoding codons 64 total codons

#

$ % &

' ( n

Equation 2.2: n, number of amino acids in peptide.

C HAPTER 3 – B IOSYNTHETIC PRODUCTION OF BIOACTIVE