02-methods.Rmd 66.9 KB
 ab604 committed Aug 17, 2019 1 2 # Methods {#methods}  mk11g11 committed Sep 16, 2019 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 {r echo=FALSE, results="hide", include=FALSE} library(grid) library(cowplot) library(tidyverse) library(ggpubr) library(readr) library(ggplot2) library(scales) library(curl) library(devtools) library(extrafont) library(magick) library(kableExtra)  ## General bacterial methods ### Transformation of *E. coli* with DNA vectors  mk11g11 committed Feb 19, 2020 22 Briefly, 25 to 50 $\mu$L of chemically competent Mach1 or DH5$\alpha$ *Escherichia coli (E.coli)* cells were combined and gently mixed with ~10 pg of plasmid DNA. The mix was left on ice for 30 minutes. Cells were placed in 42$^\circ$C water bath and after 45 seconds placed back on ice. Next, 250-500 $\mu$L of growth medium (Luria-Bertani (LB) broth) was added and cells placed in the 37 $^\circ$C shaking incubator for 45 minutes to 1 hour. The entire volume of cells was spread onto 10 cm LB-agar plate containing an appropriate antibiotic for selection. Antibiotics used for each vector selection are listed in Table \@ref(tab:antibiotics-used). Cells were spread on plates were left in the 37$^{\circ}$C incubator overnight to allow for growth of transformed cells into colonies.  mk11g11 committed Sep 16, 2019 23 24 25 26 27  {r antibiotics-used, echo=FALSE} library(kableExtra) text_tbl <- data.frame( Plasmid = c("pET26", "pBMH", "pET27b(+)", "pcDNA3.1", "PCR-8-TOPO", "pDEST"),  mk11g11 committed Feb 19, 2020 28  Antibiotic = c("Kanamycin", "Ampicillin", "Kanamycin", "Ampicillin", "Spectinomycin", "Ampicillin"))  mk11g11 committed Sep 16, 2019 29 30 31  knitr::kable(text_tbl, format = "latex", caption = 'Selection pressure for DNA plasmids used in this study.', booktabs = TRUE) %>% kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%  mk11g11 committed Oct 06, 2019 32  footnote(general = "Ampicillin used at a final concentration of 0.1 mg/mL, whereas kanamycin and spectinomycin at 0.05 mg/mL.",  mk11g11 committed Sep 16, 2019 33 34 35 36 37 38  threeparttable=T)  \newpage ### Isolation of DNA plasmid from *E. coli* ### {#miniprep}  mk11g11 committed Feb 19, 2020 39 Transformed *E.coli* colony was picked, placed in 5 mL of LB supplemented with appropriate antibiotic and incubated overnight at 37 $^{\circ}$C whilst shaking. DNA was extracted using the MiniPrep Kit (Thermo Scientific or Qiagen) following the manufacturers instructions. Centrifugation was carried out in table top centrifuge at 12 000 g. Isolated DNA was quantified in a Nanodrop UV-Vis spectophotometer.  mk11g11 committed Sep 16, 2019 40 41  ### Analytic digestion of DNA plasmids  mk11g11 committed Oct 06, 2019 42 DNA plasmids were digested with restriction enzyme(s) (Promega). The reaction mix (Table \@ref(tab:RE-reaction) was incubated at 37 $^{\circ}$C for 2-8 hours and the DNA fragments were resolved on the agarose gel (Section \@ref(electrophoresis)).  mk11g11 committed Sep 16, 2019 43 44 45 46 47 48 49 50 51  {r RE-reaction, echo=FALSE} RE_reaction <- data.frame( Remove = c("10x Buffer", "BSA", "DNA", "Enzyme", "dH$_2$O"), Remove = c("1 $\\mu$L", "0.1 $\\mu$L", "1-2 $\\mu$g", "5 units", "up to 10 $\\mu$L")) names(RE_reaction) <- NULL  mk11g11 committed Feb 19, 2020 52 knitr::kable(RE_reaction, "latex", escape = FALSE, booktabs = TRUE, caption = "Components assembled to cary out restriction enzyme reaction.") %>%  mk11g11 committed Sep 16, 2019 53 54 55 56 57 58 59 60 61 62  kable_styling(latex_options = "hold_position") # %>% kable_styling(position = "center") # use the option escape=FALSE to be able to pass greek letters to the table, booktabs = TRUE means there will only be a top and bottom, and not all borders, caption = NA (no caption, but the table will be in the middle, otherwise it is ligned to the left of the page). Note that the styling function does not work here because it is from the extra package  ## General molecular biology methods ### Amplification of DNA fragments by Polymerase Chain Reaction (PCR) #### Primer design  mk11g11 committed Feb 19, 2020 63 To enable the amplification of the DNA of interest, appropriate PCR primers were designed applying the following criteria: primers were unique to the annealing site on the designated DNA, 18 to 25 nucleotides long, guanine-cytosine content from 40 to 60 %, melting temperature from 55 to 75 $^{\circ}$C. Where possible, primers rich in guanine and cytosine at 3’ end were selected to facilitate high specificity of primer binding to the target. The primers were ordered from Eurofins Genomics, subsequently diluted in ddH~2~O to the concentration of 100 pmol/$\mu$l and stored at -20 $^{\circ}$C. Sequences of primers used in this study can be found in Table \@ref(tab:primer-seq1).  mk11g11 committed Sep 16, 2019 64 65 66 67 68  {r primer-seq1, echo=FALSE} library(kableExtra) library(dplyr) pcr_primer_seqs <- data.frame(  mk11g11 committed Feb 19, 2020 69  DNA.product = c("\\textit{Ndel-pelB-3C-SalI}", "\\textit{SalI,$\\alpha$7ECD-2GSC-NheI}", "\\textit{CHRNA7}", "\\textit{eat-2}"),  mk11g11 committed Sep 16, 2019 70 71 72 73 74 75 76 77 78 79 80 81 82 83  Primer = c("Fw: gaaggagatatacatatgaaatacctg\nRv: TAGCTTGTCGACgggcccctggaacagaacttc", "Fw: AGCTCCGTCGACtttcagcgtaaactgtacaaag\nRv: ACTAGCTAGCTTAaagcttagccgcaccacggcg", "Fw: atgcgctgctcgccgggaggcg\nRv: ttacgcaaagtctttggacacggc", "Fw: atgaccttgaaaatcgca\nRv: ttattcaatatcaacaatcgg"), Size = c("1560", "1051", "1509", "1425")) pcr_primer_seqs %>% mutate_all(linebreak) %>% kable("latex", booktabs = T, escape = F, col.names = linebreak(c("DNA product", "DNA primer", "Size\n(bp)")), caption = "DNA primers used in this study.") %>% kable_styling(latex_options = "hold_position") %>% footnote(general = "Sequences are presented in 5' to 3' direction, non-complementary nucleotides are in capital letters whereas complementary in small letters. Fw stands for forward, whereas Rv for reverse primer.", threeparttable = T)  \newpage #### PCR Protocol  mk11g11 committed Feb 19, 2020 84 PCR was performed using either Phusion High-Fidelity DNA Polymerase (Thermo Scientific) or Pfu DNA polymerase (Promega) as indicated. The components mixed and cycling conditions used are listed in Tables \@ref(tab:Phusion-pol) and \@ref(tab:Pfu-pol).  mk11g11 committed Sep 16, 2019 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148  {r Phusion-pol, echo=FALSE} library(kableExtra) library(dplyr) phusion_components <- data.frame( Component = c("Buffer", "dNTP mix", "Reverse/\nForward primer", "DNA", "Polymerase", "ddH$_2$O"),  mk11g11 committed Feb 19, 2020 149 Concentration = c("1x", "200 $\\mu$M", "500 nM", "10 ng / 50 $\\mu$L reaction", "0.5 U / 50 $\\mu$L reaction", "up to 50 $\\mu$L"))  mk11g11 committed Sep 16, 2019 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179  phusion_components %>% mutate_all(linebreak) %>% kable("latex", booktabs = T, escape = F, caption = "Components assembled for Phusion polymerase-mediated PCR reaction.") %>% kable_styling(latex_options = "hold_position")  {r Pfu-pol, echo= FALSE} library(kableExtra) library(dplyr) phusion_components <- data.frame( Component = c("Buffer", "dNTP mix", "Reverse/\nForward primer", "DNA", "Polymerase", "ddH$_2$O"),  mk11g11 committed Feb 19, 2020 180 Concentration = c("1x", "200 $\\mu$M", "500 nM", "25 ng / 50 $\\mu$L reaction", "0.25 U / 50 $\\mu$L reaction", "up to 50 $\\mu$L"))  mk11g11 committed Sep 16, 2019 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207  phusion_components %>% mutate_all(linebreak) %>% kable("latex", booktabs = T, escape = F, caption = "Components assembled for Pfu polymerase-mediated PCR reaction.") %>% kable_styling(latex_options = "hold_position")  \newpage  mk11g11 committed Feb 19, 2020 208 Thermal cycling conditions used for amplification of *C. elegans* nAChR subunit *eat-2*, whole length and the extracellular domain of human $\alpha7$ nAChR subunit as well as $pelB-HIS-MBP-3C$ sequence (sequence for expression of genes and purification of proteins from *E. coli*) are shown in Tables \@ref(tab:eat2-amplification) - \@ref(tab:human-lgd-amplification).  mk11g11 committed Sep 16, 2019 209 210 211  {r eat2-amplification, echo=FALSE} eat2_amplification <- data.frame(  mk11g11 committed Feb 19, 2020 212  Step = c("Initial denaturation", "Denaturation", "Annealing", "Extension", "Final extension"),  mk11g11 committed Sep 16, 2019 213 214 215 216 217 218 219  Duration = c("2 mins", "1 mins", "30 secs", "3 mins", "5 mins"), Temperature = c("95", "95", "51.1", "73", "73"), Number = c ("1", " ", "30", " ", "1")) eat2_amplification %>% mutate_all(linebreak) %>% kable("latex", booktabs = T, escape = F,  mk11g11 committed Feb 19, 2020 220  col.names = linebreak(c("Step", "Duration", "Temperature $^\\circ$C", "Number\nof cycles")), caption = "Thermal cycling conditions for amplification of eat-2 from pTB207 plasmid with Pfu polymerase.") %>%  mk11g11 committed Sep 16, 2019 221 222 223 224 225 226 227 228 229  kable_styling(latex_options = "hold_position")  {r CHRNA7-amplification, echo=FALSE} library(kableExtra) library(dplyr) CHRNA7_amplification <- data.frame(  mk11g11 committed Feb 19, 2020 230  Step = c("Initial denaturation", "Denaturation", "Annealing", "Extension", "Final extension"),  mk11g11 committed Sep 16, 2019 231 232 233 234 235 236 237  Duration = c("30 secs", "10 secs", "30 secs", "45 secs", "7 mins"), Temperature = c("98", "98", "gradient", "72", "72"), Number = c ("1", " ", "30", " ", "1")) CHRNA7_amplification %>% mutate_all(linebreak) %>% kable("latex", booktabs = T, escape = F,  mk11g11 committed Feb 19, 2020 238  col.names = linebreak(c("Step", "Duration", "Temperature $^\\circ$C", "Number\nof cycles")), caption = "Thermal cycling conditions for amplification of human $\\alpha$7 nAChR (CHRNA7) from pcDNA3.1 plasmid with Phusion polymerase.") %>%  mk11g11 committed Sep 16, 2019 239  kable_styling(latex_options = "hold_position") %>%  mk11g11 committed Feb 19, 2020 240  footnote(general = "gradient annealing temperatures were: 50, 51.1, 53.4, 57.2, 60.2",  mk11g11 committed Sep 16, 2019 241 242 243 244 245 246 247 248 249 250  threeparttable = T)  \newpage {r MBP-amplification, echo=FALSE} library(kableExtra) library(dplyr) mbp_amplification <- data.frame(  mk11g11 committed Feb 19, 2020 251  Step = c("Initial denaturation", "Denaturation", "Annealing", "Extension", "Final extension"),  mk11g11 committed Sep 16, 2019 252 253 254 255 256 257 258  Duration = c("3 mins", "1 mins", "30 secs", "3 mins", "5 mins"), Temperature = c("95", "95", "45", "73", "73"), Number = c ("1", " ", "35", " ", "1")) mbp_amplification %>% mutate_all(linebreak) %>% kable("latex", booktabs = T, escape = F,  mk11g11 committed Feb 19, 2020 259  col.names = linebreak(c("Step", "Duration", "Temperature $^\\circ$C", "Number\nof cycles")), caption = "Thermal cycling conditions for amplification of pelB-HIS-MBP-3C from pET26-GLIC plasmid with Pfu polymerase.") %>%  mk11g11 committed Sep 16, 2019 260 261 262 263 264 265 266 267  kable_styling(latex_options = "hold_position")  {r human-lgd-amplification, echo=FALSE} library(kableExtra) library(dplyr) humanlgd_amplification <- data.frame(  mk11g11 committed Feb 19, 2020 268  Step = c("Initial denaturation", "Denaturation", "Annealing", "Extension", "Final extension"),  mk11g11 committed Sep 16, 2019 269 270 271 272 273 274 275  Duration = c("30 secs", "10 secs", "45 secs", "45 secs", "7 mins"), Temperature = c("98", "98", "51.7", "72", "72"), Number = c ("1", " ", "35", " ", "1")) humanlgd_amplification %>% mutate_all(linebreak) %>% kable("latex", booktabs = T, escape = F,  mk11g11 committed Oct 06, 2019 276  col.names = linebreak(c("Step", "Duration", "Temperature $^\\circ$C", "Number\nof cycles")), caption = "Thermal cycling conditions for amplification of human $\\alpha$7 nAChR ligand binding domain from pBMH plasmid with Phusion HF polymerase (Thermo Scientific).") %>%  mk11g11 committed Sep 16, 2019 277 278 279 280 281 282 283  kable_styling(latex_options = "hold_position")  \newpage ### DNA electrophoresis ### {#electrophoresis}  mk11g11 committed Feb 19, 2020 284 To resolve the size of DNA samples, agarose gel electrophoresis was run using BioRad Wide horizontal electrophoresis system and PowerPac Basic Power Supply. The resolving gel was prepared by addition of agarose (0.6-1.2 % (w/v); Sigma Aldrich) to 1x TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) buffer. This mix was heated in the microwave until agarose completely melted and left on the bench to cool down to ~ 50 $^\circ$C. Subsequently, Nancy-520 DNA Gel Stain (Sigma-Aldrich) at 5 mg/mL was added in 1:1000 (v/v) dilution and the mixture was poured into the gel caster. Meanwhile, the DNA samples were prepared by mixing them with 1 % (v/v) loading dye (Blue/Orange Loading Dye, Promega). Once the gel set, the samples were loaded into wells alongside the indicated molecular weight marker. Markers variously used in this thesis include 1kb Hyperladder (Bioline), 1kb ladder (Promega) or 1 kb Plus DNA ladder (Thermo Scientific). Electrophoresis was run at 70 V until the samples were sufficiently resolved (typically 30 minutes to 2 hours) and gels imaged using Syngenta GBox.  mk11g11 committed Sep 16, 2019 285 286 287  ### DNA purification following PCR and electrophoresis Following gel electrophoresis, the band of interest was visualised under the UV light and isolated by cutting with a surgical blade.  mk11g11 committed Feb 19, 2020 288 DNA was subsequently purified using GeneJET Gel Extraction Kit (Thermo Scientific) or Gel Extraction Kit (Qiagen) following manufacturers protocols.  mk11g11 committed Sep 16, 2019 289 290 291 292   mk11g11 committed Feb 19, 2020 293 294 295  ### Ligation dependent cloning ###{#ligationdependentcloning} The DNA vector for expression of human nAChR was generated by T4 dependent ligation. PCR-amplified, gel-excised and digested with SalI and NdeI pelB-HIS-MBP-3C gene was inserted into the digested pET27 plasmid. The complementary sequences were ligated with T4 ligase (Table \@ref(tab:T4-ligase)). Next, PCR-amplified, gel-excised and digested with SalI and NheI human $\alpha7$ extracellular domain (ECD) was ligated into the digested pET27-pelB-HIS-MBP-3C. Both times reaction mixtures were incubated at room temperature for 3-4 hours and used to transform chemically competent Mach1 cells. 50-100 $\mu$L of cells were transformed with 2.5-8 $\mu$L ligation reaction mix.  mk11g11 committed Sep 16, 2019 296 297 298 299 300 301 302 303 304  {r T4-ligase, echo=FALSE} ligation_table <- data.frame( Remove = c("Ligase buffer", "Backbone DNA", "Insert DNA", "T4 DNA ligase", "ddH$_2$O"), Remove = c("1 x", "100 ng", "3:1 insert:backbone ratio", "1 unit", "up to 10 $\\mu$L")) names(ligation_table) <- NULL  mk11g11 committed Feb 19, 2020 305 kable(ligation_table, format = "latex", escape = FALSE, align = 'l', booktabs = TRUE, caption = "Components assembled to carry out ligation-dependent cloning reaction.") %>% kable_styling(position = "center", latex_options = "hold_position")  mk11g11 committed Sep 16, 2019 306 307 308 309 310  # use the option escape=FALSE to be able to pass greek letters to the table, booktabs = TRUE means there will only be a top and bottom, and not all borders, caption = NA (no caption, but the table will be in the middle, otherwise it is ligned to the left of the page)   mk11g11 committed Oct 06, 2019 311 ### Gateway cloning ###{#gatewaycloning}  mk11g11 committed Feb 19, 2020 312 Vectors for generation of *C. elegans* transgenic genes were generated by recombinant Gateway Cloning.  mk11g11 committed Sep 16, 2019 313 314 315 316 317 318 319   mk11g11 committed Feb 19, 2020 320 321 #### Generation of the entry (TOPO) vector by TA recombination ####{#adenosineoverhang} 3' adenine overhangs were added to the amplified and gel purified DNA fragment in the reaction using non-proofreading Extend Long Roche Polymerase (ThermoScientific) (Table \@ref(tab:a-overhangs-addition)).  mk11g11 committed Sep 16, 2019 322 323 324 325 326 327 328 329 330 331  {r a-overhangs-addition, echo=FALSE} gateway_cloning <- data.frame( Remove = c("Polymerase", "10 x Buffer B", "DNA", "dNTP", "ddH$_2$O"), Remove = c("5 U/20 $\\mu$L reaction", "1x", "up to 500 ng", "200 $\\mu$M", "up to 10 $\\mu$L")) names(gateway_cloning) <- NULL options(knitr.table.format= "latex")  mk11g11 committed Feb 19, 2020 332 knitr::kable(gateway_cloning, escape = FALSE, booktabs = TRUE, caption = "Addition of adenine overhangs to PCR product for entry clone generation.") %>%  mk11g11 committed Sep 16, 2019 333 334 335 336 337 338 339  kable_styling(latex_options = "hold_position") # %>% kable_styling(position = "center") # use the option escape=FALSE to be able to pass greek letters to the table, booktabs = TRUE means there will only be a top and bottom, and not all borders, caption = NA (no caption, but the table will be in the middle, otherwise it is ligned to the left of the page)   mk11g11 committed Feb 19, 2020 340 TA reaction was assembled (Table \@ref(tab:TA-reaction)), incubated at room temperature for 1 hour and 2 $\mu$L of the reaction mix was used to transform 50 $\mu$L DH5$\alpha$ chemically competent cells. DNA was isolated from transformed colonies and sequenced to ensure the correct sequence and orientation of the insert.  mk11g11 committed Sep 16, 2019 341 342 343 344 345 346 347 348 349 350  {r TA-reaction, echo=FALSE} topo_reaction_tb <- data.frame( Remove = c("PCR-8 TOPO vector", "Salt solution", "PCR product", "ddH$_2$O"), Remove = c("1 $\\mu$L", "1 $\\mu$L", "up to 500 ng", "up to 6 $\\mu$L")) names(topo_reaction_tb) <- NULL options(knitr.table.format= "latex")  mk11g11 committed Feb 19, 2020 351 knitr::kable(topo_reaction_tb, escape = FALSE, align = 'l', booktabs = TRUE, caption = "Components assembled for the generation of the entry clone for Gateway cloning.") %>%  mk11g11 committed Sep 16, 2019 352 353 354 355 356 357  kable_styling(latex_options = "hold_position") # %>% kable_styling(position = "center") # use the option escape=FALSE to be able to pass greek letters to the table, booktabs = TRUE means there will only be a top and bottom, and not all borders, caption = NA (no caption, but the table will be in the middle, otherwise it is ligned to the left of the page)   mk11g11 committed Feb 19, 2020 358 #### Generation of the expression vector by LR reaction ####{#lr-reaction-section}  mk11g11 committed Oct 06, 2019 359 LR reaction was assembled using Gateway LR Clonase II Enzyme Mix (Invitrogen) (Table \@ref(tab:LR-reaction). Reaction was incubated at room temperature for 2 hours. To inactivate the enzyme, 2 $\mu$L of proteinase K was added and the reaction mix was incubated at 37 $^\circ$C for 10 minutes. 1 $\mu$L of reaction mix was used to transform 50 $\mu$L of One Shot OmniMAX 2 T1 phage resistant cells (Invitrogen). Transformed cells were plated and grew overnight. Following, plasmid was isolated from transformed cells and subjected to sequencing to ensure successful formation of the plasmid.  mk11g11 committed Sep 16, 2019 360 361 362 363 364 365 366 367 368 369  {r LR-reaction, echo=FALSE} gateway_cloning2 <- data.frame( Remove = c("Entry clone (PCR-8-TOPO-CHANR7)", "Destination vector (pDEST-Pmyo2)", "LR Clonase II", "TE buffer (pH=8)"), Remove = c("75 ng", "75 ng", "1 $\\mu$L", "up to 5 $\\mu$L")) names(gateway_cloning2) <- NULL options(knitr.table.format= "latex")  mk11g11 committed Feb 19, 2020 370 knitr::kable(gateway_cloning2, escape = FALSE, align = 'l', booktabs = TRUE, caption = "Components assembled for the generation of recombinant vector by gateway cloning.") %>%  mk11g11 committed Sep 16, 2019 371 372 373 374 375 376 377 378 379  kable_styling(latex_options = "hold_position") # %>% kable_styling(position = "center") # use the option escape=FALSE to be able to pass greek letters to the table, booktabs = TRUE means there will only be a top and bottom, and not all borders, caption = NA (no caption, but the table will be in the middle, otherwise it is ligned to the left of the page)  ## Expression of human $\alpha7$ nAChR in *E. coli* Heterologous protein was expressed in and subsequently purified from *E. coli* (Figure \@ref(fig:purification-label)).  mk11g11 committed Feb 19, 2020 380 (ref:purifcation-fig) **The process of heterologous protein expression in *E. coli* and protein purification.** Plasmid containing gene of interest is transformed into *E. coli* cells. Transformed cells are grown in medium and the protein expression induced by addition of IPTG. Cells are subsequently harvested, re-suspended in buffer and cellular content released by sonication. The supernatant containing soluble proteins is isolated from cellular debris by centrifugation and the heterologous protein isolated using metal affinity chromatography. His tagged protein bound to Nickel^2+^ are eluted with imidazole, whereas Maltose Binding Protein (MBP) tagged proteins bound to Dextrin Sepharose beads are eluted with maltose.  mk11g11 committed Sep 16, 2019 381 382 383 384 385 386  {r purification-label, fig.cap="(ref:purifcation-fig)", fig.scap= "The process of heterologous protein expression in \\textit{E. coli} and subsequent purification.", fig.align= "centre", echo=FALSE, fig.pos = 'H'} knitr::include_graphics("fig/methods/purification-process.png")  ### Growth of transformed *E. coli* cells  mk11g11 committed Feb 19, 2020 387 Chemically competent bacterial cells (BL21(DE3)) engineered for high efficiency protein expression were transformed with the authenticated expression vector as described (Section \@ref(miniprep)). Transformed colonies were resuspended and placed in 5 mL of growth medium supplemented with the appropriate antibiotic. Seed culture was placed in the shaking incubator at 37 $^\circ$C and left to grow until OD~600nm~ of 1-2. This starter culture was used to inoculate growth medium supplemented with appropriate antibiotic in 2 L baffled flasks, to the final OD~600nm~ of 0.01-0.05. Inoculated flasks were placed in a shaking incubator at 37 $^\circ$C, 250 RPM. The following protocol was followed, unless otherwise stated: At OD~600nm~ = 0.5, the temperature was lowered to 18 $^\circ$C. When the 18 $^\circ$C culture reached an OD~600nm~ ≈ 1, 0.2 mM isopropyl $\beta$-D-1-thiogalactopyranoside (IPTG) was added and the growth continued overnight at 18 $^\circ$C.  mk11g11 committed Sep 16, 2019 388 389  ### Protein purification ### {#purification-general-methods}  mk11g11 committed Feb 19, 2020 390 *E.coli* were harvested by centrifuging the cell culture at 5000 g for 20 minutes at 4 $^\circ$C and either used immediately or stored at -20 $^\circ$C for further use.  mk11g11 committed Sep 16, 2019 391 392 393 394 395 Harvested cells were kept on ice throughout the purification procedure. Two methods of purification were tested: HIS-tag purification using Ni-NTA resin and maltose binding protein (MBP) purification with Sepharose-Dextrin Beads (GE Healthcare Life Sciences). ### HIS-tag purification #### {#his} The composition of buffers used is as follows :  mk11g11 committed Feb 19, 2020 396 Re-suspension buffer: 0.1M TRIS (pH=8), 0.15 M NaCl. Wash buffer 1: as previous. Wash buffer 2: 0.1 M TRIS (pH=8), 1 M NaCl. Wash buffer 3: 0.1 M TRIS, (pH=8), 0.15 M NaCl. Elution buffer: 0.1 M TRIS, (pH=8), 0.15 M NaCl, 0.2 M imidazole (pH=7.5)  mk11g11 committed Sep 16, 2019 397   mk11g11 committed Feb 19, 2020 398 Cells harvested from 1 L of culture medium were re-suspended in 40 mL re-suspension buffer supplemented with 2 Pierce™ Protease Inhibitor Mini Tablets (Thermo Fisher Scientific) and sonicated on ice using the following settings: power 7, pulse on: 10 seconds, pulse off: 20 seconds, total time 6 minutes. Sonicated cells were subject to 16000 g spin for 45 minutes at 4 $^\circ$C to sediment cellular debris. The supernatant was collected and spun again at 100 000 g for 1 hour at 4 $^\circ$C to separate non-soluble fraction (e.g. aggregated proteins) in the pellet from the supernatant containing soluble fraction. Supernatant was mixed with 0.5 mL of Ni-NTA resin (previously equilibrated in the resuspension buffer) and equilibrated for 1 hour or overnight at 4 $^\circ$C on the rotating tube rotator (speed 8-9). Following this incubation, the mix was decanted into a low pressure 5 mL chromatography column. Resin was washed with 10 mL of each one of the 3 washing buffers. Lastly, bound to Ni-NTA resin proteins were eluted off by addition of 5 x 0.5 mL of elution buffer. Eluted fractions were stored at 4 $^\circ$C. At each stage, a samples consisting of the pre induction (pre-I; post induction (post-I) Homogenate (H) (Whole cells), high speed supernatant (LOAD), Flow through (FT0) wash (W) and eluate (E) fractions were collected for SDS-PAGE analysis (Section \@ref(samples)).  mk11g11 committed Sep 16, 2019 399 400 401 402 403 404 405 406 407 408 409 410 411  ### Quantification of protein expression and purification  mk11g11 committed Feb 19, 2020 412 Protein content of the eluted samples was measured with NanoDrop 1000 Spectrophotometer V3.7 at 280 nM and the following parameters, as measured by Compute pI/Mw tool (http://web.expasy.org/compute_pi/): Mw (kDa) of pentameric full length protein = 420, extinction coefficient (/1000)= 132.95. Two $\mu$L of the elution buffer/buffer C were used to blank the spectrophotometer, and 2 $\mu$L of the elution fractions was used to estimate the protein concentration of the sample.  mk11g11 committed Sep 16, 2019 413   mk11g11 committed Oct 06, 2019 414 ### Analysis of protein molecular weight using denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)  mk11g11 committed Sep 16, 2019 415 416  #### Sample preparation #### {#samples}  mk11g11 committed Feb 19, 2020 417 This section described how each sample was prepared. Pre and post induction whole cell samples: 1 mL of *E. coli* cell culture was taken, spun down in tabletop centrifuge at max speed for 5 minutes. Supernatant was discarded, pellet re-suspended in 150 $\mu$L dH~2~O. Total protein content of 2 $\mu$L samples were measured with NanoDrop by measuring protein absorbance at 280 nm. Seventy mg/ml of protein was loaded onto a gel in each sample. Cell lysate: following sonication, 30 $\mu$L of cell lysate was taken. Cell-debris and supernatant: 30 $\mu$L of cell lysate sample taken, spun down in tabletop centrifuge for 10 mins at maximum speed, at 4 $^\circ$C. supernatant was pipetted into another micro centrifuge (labelled supernatant) tube whereas pellet re-suspended in 30 $\mu$L dH~2~O (labelled whole-cell). Supernatant and Pellet samples: 50 $\mu$L of cell lysate spun down in tabletop centrifuge for 10 mins at maximum speed, at 4 $^\circ$C. Thirty $\mu$L of the supernatant taken, spun down in ultracentrifuge at 100 000 g, at 4 $^\circ$C for 1 hour. Supernatant was pipetted into another microcentrifuge tube (ultra-supernatant) whereas debris was re-suspended in 30 $\mu$L of dH~2~0. The same volumes of cell-lysate, cell-debris, supernatant, ultra-supernatant, ultra-pellet and flow-through were loaded onto SDS-PAGE gels. Protein samples were mixed with sample buffer (2 % SDS, 2 mM DTT, 4 % glycerol, 0.04 M Tris pH = 6.8, 0.01 % bromophenol blue) and boiled for 5 – 10 minutes. Next, 4 – 10 $\mu$L samples were loaded onto 8 – 12 % acrylamide SDS-PAGE gel alongside 4 $\mu$L of Protein Marker PageRulerTM Prestained/Unstained Protein (Thermo Fisher Scientific).  mk11g11 committed Sep 16, 2019 418 419 420 421  #### Gel electrophoresis ##### Gel preparation ####{#gelprep}  mk11g11 committed Feb 19, 2020 422 Protein samples were subject to denaturing SDS-PAGE and stained with the Commassie stain to visualise and estimate the size of protein species. SDS-PAGE constituted from a stacking gel (5 % acrylamide/bisacrylamide, 0.72 M Tris pH = 8.4, 0.025 % ammonium persulfate, 0.4 % TEMED, 0.1 % SDS) casted over a resolving gel (12 % acrylamide/bisacrylamide, 1 M Tris pH = 8.4, 0.06 % ammonium persulfate, 0.13 % TEMED, 0.1 % SDS).  mk11g11 committed Sep 16, 2019 423   mk11g11 committed Feb 19, 2020 424 BioRad electrophoresis apparatus was used. Electrophoresis chamber was filled with the running buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS) and the electrophoresis proceeded at 80 V for 1 hour and then 120 V for 2 hours. The gel was either stained to visualise all proteins present in samples, or used in Western blot to detect the presence of a specific protein.  mk11g11 committed Sep 16, 2019 425   mk11g11 committed Sep 29, 2019 426 #### Coomassie staining and imaging ####{#coomassiestaining}  mk11g11 committed Feb 19, 2020 427 Following SDS-PAGE electrophoresis, the stack and resolving gel were incubated in fixing buffer (10 % acetic acid, 40 % ethanol, 50 % dH~2~O) placed on an oscillating platform for at least 1 hour to remove background staining. The fixed gel was then washed with dH~2~O and incubated with Coomassie stain (14 mg of Coomassie Blue R-250 (ThermoScientific) /L of dH~2~O) overnight. The gel was de-stained by incubation with dH~2~O for at least 8 hours and imaged with Gel Doc^TM^ XR+ (Bio-Rad).  mk11g11 committed Sep 16, 2019 428   mk11g11 committed Oct 06, 2019 429 ### Analysis of protein molecular weight using one-dimensional non-denaturing polyacrylamide gel electrophoresis  mk11g11 committed Sep 16, 2019 430 431  #### Sample preparation.  mk11g11 committed Feb 19, 2020 432 Two protein eluate samples were collected from the immobilized metal affinity chromatography (IMAC) and mixed with sample buffer (4 % glycerol, 0.04 M Tris pH = 6.8, 0.01 % bromophenol blue). One sample was boiled for 5 minutes to denature proteins, whereas the other was not. Next, 4 – 10 $\mu$L prepared samples were loaded onto 12 % nondenaturing polyacrylamide gel, alongside 4 $\mu$L of 1 mg/mL of bovine serum albumin, which served as a marker.  mk11g11 committed Sep 16, 2019 433 434  ##### Gel preparation  mk11g11 committed Feb 19, 2020 435 Nondenaturing gel (12 % acrylamide/bisacrylamide, 1 M Tris pH = 8.4, 0.06 % ammonium persulfate, 0.13 % TEMED) was used to resolve the size of proteins.  mk11g11 committed Sep 16, 2019 436 437  ##### Gel electrophoresis  mk11g11 committed Feb 19, 2020 438 BioRad electrophoresis apparatus was used. Electrophoresis chamber was filled with the running buffer (25 mM Tris, 192 mM glycine) and the electrophoresis proceeded at 80 V for 1 hour and then 120 V for 2 hours. The gel was stained and imaged as described in Section \@ref(coomassiestaining)  mk11g11 committed Sep 16, 2019 439   mk11g11 committed Feb 19, 2020 440 ### Western blots ###{#western}  mk11g11 committed Sep 16, 2019 441   mk11g11 committed Feb 19, 2020 442 443 #### Protein transfer Resolved protein are transferred to polyvinylidene difluoride (PVDF). Membrane cut to the size of the resolving gel was equilibrated for 15 minutes to 1 hour in the transfer buffer (12.1 g Tris, 57.6 g glycine, 800 mL methanol in total volume of 4 L) then washed with dH~2~O followed by methanol. Freshly run polyacrylamide gel was placed on top of the submerged in transfer buffer sponge, 2 x filter paper and PVDF membrane stack and covered with 2 x filter paper. Assembled transfer mount with the gel and PVDF membrane was placed in the BioRad Mini Trans-Blot Module which was in turn inserted into Mini-PROTEAN Tetra Cell tank. The tank was filled with transfer buffer and the protein transferred from the gel onto the membrane at 100 V constant voltage for for 1 - 3 hours at 4 $^\circ$C.  mk11g11 committed Sep 16, 2019 444 445  #### Antibody binding ####{#abs}  mk11g11 committed Feb 19, 2020 446 Transfer of his-tagged human $\alpha7$ nAChR ECD-chimera protein were detected using 1 mg/mL monoclonal mouse anti-Hexa-His primary antibodies (Thermo Fisher Scientific) and 1mg/mL IRDye® 680RD Goat anti-Mouse IgG (Li-Cor) used at 1 in 1000 dilution. PVDF membrane was incubated for at least 1 hour in blocking, primary and secondary antibody buffer (Table \@ref(tab:WB-buffers)). To remove residual solution, three 10-minute-long washes were carried out in-between and after last incubations.  mk11g11 committed Sep 16, 2019 447 448 449 450 451 452 453 454 455 456 457  {r WB-buffers, echo=FALSE, fig.pos = 'H'} wb_bfrs <- data.frame( A = c("Washing buffer", "Blocking buffer", "Primary Antibody buffer", "Secondary Antibody buffer"), Remove = c("1 x phosphate buffered saline (PBS), 0.05 $\\%$ TWEEN", "1 x PBS, 0.05 $\\%$ (v/v) TWEEN 20 (BioRad), 5 $\\%$ BSA", "5 mL Blocking buffer, 5 $\\mu$L primary antibody", "5 mL blocking buffer, 5 $\\mu$L secondary antibody")) # note that the percentage sign also has to be in a math mode names(wb_bfrs) <- NULL options(knitr.table.format= "latex")  mk11g11 committed Feb 19, 2020 458 knitr::kable(wb_bfrs, escape = FALSE, align = 'l', booktabs = TRUE, caption = "Composition of buffers used for Western blotting.") %>%  mk11g11 committed Sep 16, 2019 459 460 461 462  kable_styling(latex_options = "hold_position")# use the option escape=FALSE to be able to pass greek letters to the table, booktabs = TRUE means there will only be a top and bottom, and not all borders, caption = NA (no caption, but the table will be in the middle, otherwise it is ligned to the left of the page)   mk11g11 committed Feb 19, 2020 463 #### Western blot imaging  mk11g11 committed Oct 06, 2019 464 Immunodecorated PVDF were imaged using Odyssey imaging system (Li-Cor Biosciences). Images in the 800 nm channel detects protein bands tagged by the IRDye 800CW secondary antibody. This was cross referenced to images scanned in the 700 nm to detect protein ladder bands and 800 nm channels.  mk11g11 committed Sep 16, 2019 465 466  #### Gel filtration  mk11g11 committed Oct 06, 2019 467 Protein purified and eluted with the Ni-NTA resin were subject to size exclusion chromatography with GE Healthcare Superdex^TM^ 200 10/300GL column with the separation range between 10 and 600 kDa. This methods allows for the molecular weight assessment and separation of proteins present in the sample based on their mobility through the resin-filled column.  mk11g11 committed Sep 16, 2019 468 469  #### Sample preparation  mk11g11 committed Oct 06, 2019 470 Sample prepared with VIVASPIN20 column with the cut off point of 30 kDa (Sartorius) by spinning down in a centrifuge at 36 000 RPM at 4 $^\circ$C.  mk11g11 committed Sep 16, 2019 471 472  #### Buffers  mk11g11 committed Feb 19, 2020 473 Buffer used : 0.1M TRIS (pH=8), 0.15 M NaCl degassed and ddH~2~0 degassed.  mk11g11 committed Sep 16, 2019 474 475  #### Calibration of the column ####{#calibration}  mk11g11 committed Feb 19, 2020 476 To estimate the size of proteins present in the sample, standard curve was generated. Four proteins were selected as protein standards: trypsin of 23.3 kDa, chicken serum albumin of 47.5 kDa, bovine serum albumin of 66.5 kDa and dextrin which forms large aggregates. These aggregates are larger than the column capacity, therefore dextrin serves as void. Solutions of 3 mg/mL of trypsin, chicken and bovine serum albumin were prepared and 1 mg/mL of dextrin. Protein solutions were injected into the column one at the time at a flow rate of 0.4 mL/ min. The eluted volume at which peak position as a function of volume eluted was noted for each protein. All peak positions were normalised to the position of the void (dextrin) (Figure \@ref(fig:protein-standard-label)). Linear regression line was plotted of the log protein size (kDa) as a function of normalised volume eluted (Figure \@ref(fig:standard-curve-gel-filtration-label). The standard equation of the line was derived: y = - 0.1738 * peak position + 2. 776.  mk11g11 committed Sep 16, 2019 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494  \newpage {r, echo=FALSE, fig.pos = 'H'} text_tbl2 <- data.frame ( Protein = c("Blue dextran", "Bovine Serum Albumin", "Chicken Serum Albumin", "Trypsin"), Mwt = c("NA", "66.45", "47.29", "23.30"), log = c("NA", "1.82", "1.68", "1.37"), Peak = c("9.48", "14.73", "16.38", "17.23"), Nomalised = c("0", "5.25", "6.90", "7.75")) text_tbl2 %>% mutate_all(linebreak) %>% kable("latex", booktabs = T, escape = F, col.names = linebreak(c("Protein", "Mwt (kDa)", "log Mwt", "Peak position\n(mL)", "Normalised\npeak position"))) %>% kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position")   mk11g11 committed Feb 19, 2020 495 (ref:protein-standard) **Gel filtration of protein markers.** Data derived from the spectra and normalised to the dextran void.  mk11g11 committed Sep 16, 2019 496   mk11g11 committed Sep 29, 2019 497 {r protein-standard-label, fig.cap = "(ref:protein-standard)", fig.scap = "Gel filtration of protein markers.", fig.align = 'center', echo=FALSE, fig.pos = 'H'}  mk11g11 committed Sep 16, 2019 498 499 500 501 502 503 504 505 506 507 508 509 510 knitr::include_graphics("fig/methods/gel-filtration-spectra.png")  \newpage ## *C. elegans* methods ### *C. elegans* strains Wild type strain: N2 (Bristol) Mutant strains:  mk11g11 committed Feb 19, 2020 511 CB7431 (genotype *bus-17, (allele br2)X.* ; outcrossed x4  mk11g11 committed Sep 16, 2019 512   mk11g11 committed Feb 19, 2020 513 AD465 (genotype *eat-2, (allele ad465)II.* ; outcrossed x0  mk11g11 committed Sep 16, 2019 514   mk11g11 committed Feb 19, 2020 515 FX863 (genotype *acr-7, (alleletm863)II.* ; outcrossed x0  mk11g11 committed Sep 16, 2019 516 517 518 519 520 521 522 523 524 525  Transgenic strains: eat-2 (ad465) II Ex; [pDESTgcy32 (Pmyo-3::GFP)] eat-2 (ad465) II Ex; [[pDESTgcy32 (Pmyo-2::CHRNA7)]; [pDESTgcy32 (Pmyo-3::GFP)]] eat-2 (ad465) II Ex; [[pDESTgcy32 (Pmyo-2::EAT-2); [pDESTgcy32 (Pmyo-3::GFP)]] ### *C. elegans* culture  mk11g11 committed Feb 19, 2020 526 *C. elegans* strains were cultured at 20 $^\circ$C on the nematode growth medium (NGM) [@brenner1974] and fed with OP50 strain of *E. coli*. Worms were picked with a platinum wire.  mk11g11 committed Sep 16, 2019 527   mk11g11 committed Feb 19, 2020 528 529 ### Preparation of *C. elegans* plates ### {#plates} NGM was prepared weekly in 4 or 8 L batches as described: 2 % agar (w/v), 0.25 % peptone (w/v), 50 mM NacL (w/v) in dH~2~0. The components were autoclaved and cooled to 55 $^\circ$C, then 1 mM MgSO~4~, 1 mM CaCl~2~, 1 mM K~2~HPO~4~ and 0.1 % cholesterol were added. 10 mL NGM portions were poured into 5.5 cm Petri dishes with a peristaltic pump. Once solidified, NGM were seeded with 50 $\mu$L of OP50. OP50 was applied in the middle of the plate, creating a round food patch for *C. elegans* to feed on. Prepared plates were left overnight to allow bacteria growth.  mk11g11 committed Sep 16, 2019 530   mk11g11 committed Feb 19, 2020 531 532 ### Maintanance and preparation of *E. coli* OP50 Fresh stock of OP50 plates were prepared at monthly intervals. A single colony was picked from the OP50 stock plate and placed in 10 mL of LB. Following overnight growth, cells were streaked on a plate and allowed to form colonies by incubation at 37 $^\circ$C.  mk11g11 committed Sep 16, 2019 533   mk11g11 committed Feb 19, 2020 534 535 ### *E. coli* OP50 culture To prepare OP50 culture, a single colony was picked from the OP50 stock plate and placed in 10 mL of LB. Bacterial culture was grown in a shaking incubator at 37 $^\circ$C until OD~600nm~ reached 0.6 to reach the exponentially growing phase. Cultures were stored at 4 $^\circ$C for up to 2 week and used to seed NGM plates.  mk11g11 committed Sep 16, 2019 536 537  ### General *C. elegans* methods  mk11g11 committed Feb 19, 2020 538 All experiments, with the exception of the development assay, were performed on young hermaphrodite adults (L4 + 1 day). Drugs and reagents were purchased from Sigma Aldrich, unless otherwise stated. Behavioral observations were made using a binocular microscope, unless otherwise stated. Results are expressed as mean $\pm$ SEM of ‘N’ determinations. Graph generation and measurement of EC~50~ or IC~50~ were performed in GraphPad (version 6.07). Concentration response curves were fitted into nonlinear regression sigmoidal dose-response (three parameter logistic) equation [@hill1910].  mk11g11 committed Sep 16, 2019 539 540  ### Drug stocks  mk11g11 committed Feb 19, 2020 541 5-HT was used in form of serotonin creatinine sulfate monohydrate, ampicillin in form of sodium salt, whereas nicotine was in the form of hydrogen tartrate salt. Stock concentration of FITC-alpha-bungarotoxin (FITC-$\alpha$-Bgtx) at 500 $\mu$g/ml was made in ddH~2~O. Thiacloprid and clothianidin were dissolved in 100 % dimethyl sulfoxide (DMSO). Nitenpyram and nicotine stocks were prepared by dissolving drugs in dH~2~0 and diluted to the indicated final concentrations. Working concentration of 100 $\mu$g/mL FITC-$\alpha$Bgtx was prepared and stored at 4 $^\circ$C for up to 2 weeks. The solution was span down briefly before use to pellet aggregates. Drugs were stored at -18 $^\circ$C for long term storage (>1 month). Once defrosted, they were used within 2 weeks or discarded. Nitenpyram stock was made immediately prior to the experiment and protected from light using foil to prevent photo-degradation. Buffers used for the behavioral assays in liquid were supplemented with 0.1 % (w/v) Bovine Serum Albumin (BSA), which prevents worms from sticking to the bottom of the experimental plate. Therefore, M9 and Dent’s solution refer to buffers supplemented with BSA, unless otherwise stated.  mk11g11 committed Sep 16, 2019 542 543  ### Effects of drugs on intact *C. elegans* locomotion and feeding behavior upon acute exposure. {#liquidassay}  mk11g11 committed Feb 19, 2020 544 545 546 All assays were performed in M9 medium. M9 buffer composition is (g/litre): 6 g Na~2~HPO~4~, 3 g KH~2~PO~4~, 5 g NaCl, 0.25 g MgSO~4~.H~2~O. Worms were exposed to varying indicated concentrations of nicotine or neonicotinoids for a maximum period of 2 hours. The effects of these compounds on locomotion and feeding was scored. 10 x stock concentrations nicotine, nitenpyram and 5-HT were added to the assay to give the indicated final concentration. To keep the concentration of DMSO below the concentration that have known effects (data not shown) the stocks of thiacloprid and clothianidin in 100 % DMSO were used in 1 in 200 (0.5 %) dilution and mixed vigorously with buffer.  mk11g11 committed Sep 16, 2019 547   mk11g11 committed Sep 29, 2019 548 #### Effects of drugs on of intact *C. elegans* in liquid ####{#thrashing}  mk11g11 committed Feb 19, 2020 549 Whilst in liquid, worms exhibit rhythmical swimming-like behaviour known as thrashing. A single thrash was defined as a complete bend in the mid-point of the body. Experiments were performed in a 24-well plate filled with 450 or 497 $\mu$L buffer. 50 $\mu$L nicotine/ nitenpyram/vehicle or 2.5 $\mu$L thiacloprid/clothianidin was added to the final volume of 500 $\mu$L to achieve final desired concentration.  mk11g11 committed Sep 16, 2019 550   mk11g11 committed Feb 19, 2020 551 Worms were picked off the food and transferred to the experimental arena. After 5 minutes of acclimatization to allow recovery from mechanical transfer, the first thrashing count was performed (time 0). This provided baseline thrashing for each worm. Only thrashing worms were included in the analysis. After estimation control thrashing wells were supplemented with drug / vehicle. For experiments with 1.5 mM thiacloprid, worms were transferred in a small volume of liquid (~2 $\mu$L) from the control to the experimental well by pipetting. This method was adopted due to drug’s limited solubility. It must be noted that after a period of about 40 minutes, 1.5 mM thiacloprid began to visibly precipitate. Measurements were taken for 30 seconds typically at time points: 10, 30, 40, 60 and 120 post-addition of the drug/drug vehicle. At least three independent repeats for each condition were carried out. The number of worms in each experiment varied from 2 to 6.  mk11g11 committed Sep 16, 2019 552   mk11g11 committed Feb 19, 2020 553   mk11g11 committed Sep 16, 2019 554   mk11g11 committed Feb 19, 2020 555 556 557   mk11g11 committed Sep 16, 2019 558 559  #### Onset of paralysis {#onset}  mk11g11 committed Feb 19, 2020 560 The trashing assay was performed as described above but the time interval between measurements was reduced from 30 seconds to every 2 minutes for the first 10 minutes. Worms were exposed to drug concentrations which induced paralysis in the thrashing experiment. That is wild-type worms were submerged in 100 mM nicotine, whereas *bus-17* in 25 mM nicotine, 50 mM nitenpyram or 1.5 mM thiacloprid. These concentrations were achieved by addition of 100 $\mu$L nicotine stock or vehicle into 900 $\mu$L buffer, 5 $\mu$L of thiacloprid/clothianidin stock or vehicle into 995 $\mu$L buffer or 10 $\mu$L nitenpyram or vehicle into 90 $\mu$L buffer. Twelve well plates were used. The protocol was followed as described in Section \@ref(thrashing), but after a period of acclimatisation, worms were transferred from control to the experimental well by pipetting.  mk11g11 committed Sep 16, 2019 561 562  #### Recovery from drug-induced paralysis  mk11g11 committed Feb 19, 2020 563 564 The recovery assay was designed to determine if and how quickly worms recovered from drug-induced paralysis. Twenty four-well plates were used and worms assayed in a total volume of 500 $\mu$L (Section \@ref(thrashing)) with the exception of nitenpyram experiment in which 25 $\mu$L nitenpyram stock or vehicle was added to 225 $\mu$L buffer to give a final volume of 250 $\mu$L. Following the initial thrashing count (Section \@ref(thrashing)), worms were transferred to drug concentrations inducing paralysis. Worms were incubated in nicotine for 20 minutes, thiacloprid or nitenpyram for 1 hour - that is until a steady state inhibition or full paralysis (time point 0). Subsequently, worms were transferred to a wash well to observe the recovery and thrashing was counted at 10, 30, 60, 90, 120 and 150 minutes. Alongside this test group, a positive and a negative control experiments were carried out. For the positive control, worms were transferred from buffer to drug containing medium. For a negative control, worms were transferred from buffer to buffer containing a drug solvent and back to the buffer.  mk11g11 committed Sep 16, 2019 565 566 567  #### Effects of drugs on pharmacologically induced pharyngeal pumping of intact *C. elegans* #### {#pumping}  mk11g11 committed Feb 19, 2020 568 Pharyngeal pumping assay was employed to determine the effects of compounds on the pharynx - a feeding organ of worms. Pharyngeal pumping is mediated by three main muscular anatomical structures (the corpus, anterior isthmus and the terminal bulb) which contract and relax to suck and push in the food. This activity is coupled with a movement of the grinder - a structure responsible for crushing the food into smaller particles so it can be passed down into the intestine. Therefore, to score this behaviour, the number of grinder movements per minute was counted (where forward lateral movement of the grinder and its return to the resting place was counted as 1). In this experiment, worms were assayed in the presence of the pharyngeal stimulant 5-HT. In liquid the presence of 5-HT causes immobility and stimulate pharyngeal pumping. Experiments were performed in a 24-well plate in a total volume of 500 $\mu$L (or in 250 $\mu$L for nitenpyram). Worms were picked off the food and placed in a well containing buffer. To paralyse worms and stimulate their pharyngeal pumping, 5-HT was added from stock to a final concentration of 10 mM. After 30 minutes, the 5-HT stimulated pump rate was measured. Following, the treatment/solvent was added and the effects on pumping were recorded 30 minutes later. Data was displayed in pumps per second (Hz). Data collection was carried out in collaboration with Amelia Lewis.  mk11g11 committed Oct 06, 2019 569 570  #### Effects of drugs on *C. elegans* size  mk11g11 committed Feb 19, 2020 571 572 To determine the effects of drug exposure on worms size, *bus-17* worms were submerged in 1 mL of buffer, 50 mM nicotine, 50 mM nitenpyram, 1.5 mM thiacloprid or 2.5 mM clothianidin or vehicle control (dilutions described in Section \@ref(onset)) in 12 well plates. Four hours later, worms were transferred by pipetting onto 2 % agarose pads and immobilised with 6 $\mu$L of 10 mM sodium azide. Images were taken immediately using Nikon Eclipse Microscope. Size of worms was determined in ImageJ. The scale was set using a graticule and length measured from the tip of the tail to the tip of the head with the freehand function. For improved accuracy, three measurements of each worm were taken and an average was derived.  mk11g11 committed Sep 16, 2019 573   mk11g11 committed Oct 06, 2019 574 ### Effects of drugs on intact *C. elegans* behaviour upon 24-hour exposure ###{#onplateassay}  mk11g11 committed Feb 19, 2020 575 On-plate assays were carried out to determine the effects of prolonged drug exposure on *C. elegans* behaviour. Worms were placed on NGM plates containing the indicated drug / drug vehicle and a food source in form of *E. coli* OP50 patch. All drugs were added to the NGM at 1 in 200 dilution.  mk11g11 committed Sep 16, 2019 576 577  ### Plate preparation  mk11g11 committed Feb 19, 2020 578 579 NGM was prepared as described in Section \@ref(plates). Fifty $\mu$L of drug solution at appropriate concentration was added to 10 mL of molten NGM at approx 50 $^\circ$C and mixed by gentle inversion. 3 mL of such mix was placed in each of the three successive wells of a 6-well plate (Figure \@ref(fig:on-plate-assay-method)). The medium was left overnight to solidify. One well was then seeded with 50 $\mu$L of OP50 culture, whereas the other two wells remained unseeded. This provided an experimental arena with the food on, the cleaning well and the experimental arena containing no food. In parallel, control plates containing drug solvent (water or 0.5 % DMSO) were prepared. Due to heat instability, nitenpyram plates were prepared by pipetting 50 $\mu$L of drug solution onto solidified 3 mL NGM. The plates were left overnight to enable diffusion of the compounds into the solid agar. The appropriate well was then seeded. Nitenpyram-containing plates were covered with aluminium foil at all times, to prevent photodegradation.  mk11g11 committed Sep 16, 2019 580   mk11g11 committed Feb 19, 2020 581 (ref:on-plate-assay-fig) **Diagram of the 24-hour “on-plate” assay arena.** Drug or drug solvent was incorporated into the NGM and poured into rows of a 6-well plate. Wells in the first column were seeded with the OP50. Two to four L4 + 1 worms were placed on the experimental arena containing food source. After 24 hours, pumping rate on food and the number of eggs laid per worm were counted. Following, worms were transferred to the cleaning well and left for 5-10 minutes to remove the residual food. Worms were then transferred to the experimental arena containing no food source. After period of acclimatisation (5-10 minutes), their locomotion on food was measured by counting body bends.  mk11g11 committed Sep 16, 2019 582 583 584 585 586 587 588 589  {r on-plate-assay-method, fig.cap= "(ref:on-plate-assay-fig)", fig.scap="Diagram of the 24-hour “on-plate” assay arena.", fig.align='center', echo=FALSE, fig.pos = 'H'} knitr::include_graphics("fig/methods/on_plate_experiments.jpg")  #### Experimental protocol  mk11g11 committed Feb 19, 2020 590 591 592 593 594 595 596 597 598 Four L4 + 1 worms were picked off food from the culture plate and placed in the first well of the experimental plate containing treatment or solvent. Twenty four hours later, the following behaviours were scored: 1. Pharyngeal pumping on food (feeding behaviour): a pump was defined as described previously (Section \@ref(pumping)). Only worms present on the food lawn were included in analysis. 2. Egg-laying: The number of eggs and larvae was counted to derive the total number of eggs laid over the period of 24-hours. The total value was divided by a number of worms present on a plate. Should a worm disappear from the experimental arena, the results were not included in the analysis. 3. Body bends is the measure of locomotory ability of worms on solid medium. A single body bend was defined as a bend of the below-the-head portion of the body and counted for a period of 1 minute in the absence of food. 4. Egg-hatching: After 24-hour exposure, adults were removed from the plate leaving the eggs and the progeny behind. 24-hours later, the number of unhatched eggs present on the plate was counted. This was expressed as a % of eggs hatched (formula used: 100-(number of unhatched eggs*100/total number of eggs laid).  mk11g11 committed Sep 16, 2019 599 600 601 602 603  ### Effects of drugs on development of *C. elegans* upon long term (days) exposure #### Development assay Experiments were carried out in 12 well plate containing drug or solvent -incorporated and seeded-NGM (prepared as described previously).  mk11g11 committed Feb 19, 2020 604 Six to twelve young adult hermaphrodites were placed on drug/solvent containing OP50 NGM plates. They were left on a plate for 1 hour to lay eggs, and removed from the plate, leaving the progeny behind. The number of worms in each developmental stage was counted at time points: 24, 30, 48, 54, 72, 80, 96, 120, 144, 168 and 192 hours. Larval stages were scored by following size/vulva/eggs present criteria, described by @karmacharya2009 and shown in Figure \@ref(fig:development-method). If necessary, worms were viewed at higher magnification using Nikon Eclipse E800 microscope. Results were represented as mean % worms in each developmental stage.  mk11g11 committed Sep 16, 2019 605   mk11g11 committed Feb 19, 2020 606 (ref:dev-method) ***C. elegans* developmental stages.** Images showing all 4 larval stages of *C. elegans***. L1 are the smallest worms on the plate. L2 are slightly bigger, L3 are bigger still, more mobile and have a pre-vulvar space. L4 has a visible vulva, whereas adults had eggs present in their uterus. The same magnification was used to capture all images, thus scale bar in the top left image applied to all images. Scale bar = 1mm.  mk11g11 committed Sep 16, 2019 607 608 609 610 611 612 613 614  {r development-method, fig.cap= "(ref:dev-method)", fig.scap= "\\textit{C. elegans} developmental stages. ", fig.align='center', echo=FALSE, fig.pos = 'H'} knitr::include_graphics("fig/methods/developmental_stages_annotated.jpg")  ### Effects of drugs on *C. elegans* pharyngeal pumping in dissected head preparation.  mk11g11 committed Oct 06, 2019 615 #### Dissection of worms to remove the cuticular barrier ####{#cuthead}  mk11g11 committed Feb 19, 2020 616 L4 + 1 worms were picked off food and placed in 3.5 cm Petri dish filled with 3 mL Dent's saline. Heads containing pharyngeal musculature and nerves is separated from the rest of the body (Figure \@ref(fig:cut-head-image)). By doing so, the cuticular barrier is removed and a portion of the pharynx is exposed to the external solution. The pharynx in cut-head preparation retains its function. It pumps at an average rate of 0.13 Hz over the period of 120 minutes (Figure \@ref(fig:cut-head-ctr-label)). Pumping was defined as described previously (Section \@ref(pumping)), counted for a period of 30 seconds and expressed in Hz. Only worms pumping at rate >0 were used in experiments.  mk11g11 committed Sep 16, 2019 617 618  #### Experimental arena  mk11g11 committed Feb 19, 2020 619 Cut heads were placed in a 12-well plate filled with 1 mL of Dent's saline (glucose 1.8 g, Hepes 1.2 g, NaCl 8.2 g, KCl 0.4 g, CaCl~2~ 0.4 g, MgCl~2~ - 1 mL at 1 M, pH adjusted to 7.4 with 10 M NaoH, 0.1 % BSA (w/v), made daily) with drug solution or vehicle. 100 $\mu$L or nicotine or 5-HT was added to 900 $\mu$L of buffer to achieve desired concentration of the drug. Clothianidin and thiacloprid were used at a 1 in 1000 dilution to keep the DMSO concentration at 0.1 % (v/v). Therefore, 1 $\mu$L of drug stock was added to 999 $\mu$L of buffer. Nitenpyram experiments were performed by addition of 10 $\mu$L of drug stock to 90 $\mu$L of buffer.  mk11g11 committed Sep 16, 2019 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652  (ref:cut-head) **Dissected worm preparation.** The pharynx was liberated from the rest of the body by cutting with a surgical blade just under the terminal bulb whilst viewing under the binocular microscope. {r cut-head-image, fig.cap= "(ref:cut-head)", fig.scap= "Dissected worm preparation.", fig.align='center', echo=FALSE, fig.pos = 'H'} knitr::include_graphics("fig/methods/WHOLE_AND_CUT_HEAD_2.png")  (ref:cut-head-ctr) **Pharyngeal pumping of dissected *C. elegans* in liquid.** Cut heads were placed in Dent's saline and the pharyngeal pumping was counted over time. Measurements were made by visual observations, counted for 30 seconds and expressed in Hz. Data are $\pm$ SEM collected over $\ge$ 2 observations; number of replicates $\ge$ 4. For comparison, the average pharyngeal pumping in the presence of 1 $\mu$M 5-HT is shown is dashed purple line. {r cut-head-ctr-label, fig.cap="(ref:cut-head-ctr)", fig.scap= "Pharyngeal pumping of dissected \\textit{C. elegans*} in liquid.", fig.align='center', echo=FALSE, message=FALSE, fig.pos = 'H', warning = FALSE} #read in cut head data cut_head <- readRDS("Analysis/Data/Transformed/cut_head/summary_data") ctr_cut_head_plot <- cut_head %>% filter(Experiment==12) %>% filter(Conc==0) %>% group_by(Time) %>% ggplot(aes(Time, mean_readout, group=Conc)) + geom_line() + geom_point() + geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se)) + ylim(0, 5) + scale_x_continuous(breaks = seq(0, 130, by = 20)) + ylab("Pumping(Hz)") + xlab("Time (minutes)") + theme(text=element_text(size=12, family="sans")) + ggsave("fig/results3/raw-images/liquid_basal.pdf", width = 15, height = 8, units = "cm") knitr::include_graphics("fig/results3/liquid_basal_modified.png")  ### Stimulatory effects of drugs on pharyngeal pumping of dissected *C. elegans*  mk11g11 committed Oct 06, 2019 653 #### Effects of 5-HT ####{#cuthead-5ht}  mk11g11 committed Feb 19, 2020 654 Following dissection, the heads were placed in Dent's solution. After 5 minutes the initial count of pharyngeal pumping was made and heads were transferred to a drug containing well. Pharyngeal pumping was estimated at 10, 20, 30, 60 minutes after being placed in the drug. Heads were transferred to Dent’s solution for recovery and pumping measured 30 minutes later (90 minutes after starting the measurements). As a negative control, worms were incubated in buffer throughout the duration of the experiment.  mk11g11 committed Sep 16, 2019 655 656  #### Effects of neonicotinoids and nicotine  mk11g11 committed Feb 19, 2020 657 Experiments were set up as described above (Section \@ref(cuthead-5ht)) but the time points were: 0 (Dent's), 2, 5, 10, 15, 20, 30, 45, 60 65, 75, 90 and 120 (treatment) and 130 (recovery). As a control, cut heads were incubated with a 5-HT concentration eliciting maximal response. That is 1 $\mu$M for wild-type N2 worms and 50 $\mu$M for *eat-2* mutant worms.  mk11g11 committed Sep 16, 2019 658 659 660  ### Inhibitory effects of drugs on pharmacologically induced pharyngeal pumping of dissected *C. elegans* The effects of compounds on 5-HT stimulated pharyngeal pumping was tested.  mk11g11 committed Feb 19, 2020 661 Cut heads were exposed to 1 $\mu$M 5-HT for 10 minutes to stimulate pumping. Following this, they were transferred to a well containing 5-HT and the indicated treatments. Pharyngeal pumping was measured before and 10, 20, 30, and 50 minutes after transfer into the 5-HT plus treatment incubation. To probe for recovery, heads were placed in 1 $\mu$M 5-HT and the pump rate 5, 10 and 30 minutes after being transferred into recovery (that is 55, 65 and 80 minutes after the start of the experiment) was recorded. As a control, heads were exposed to 5-HT plus solvent throughout the duration of the experiment.  mk11g11 committed Sep 16, 2019 662 663 664 665 666  ### Extracellular recording from the pharynx of cut head preparation of *C. elegans* Cut heads were prepared (Section \@ref(cuthead)) and transferred to the experimental arena by pipetting. Extracellular recordings were made with an electropharyngeogram (EPG) technique (Figure \@ref(fig:EPG-setup-method)). #### Preparation of a microelectrode  mk11g11 committed Oct 06, 2019 667 Non-filamented borosillicate capillary tube (Havard apparatus) with outer diameter (OD) of 1.5 mm and internal diameter (ID) of 0.1 mm was pulled with a Narishige puller (model PC:10). The puller was set at 98.2 $^\circ$C for step 1 and 72.8 $^\circ$C for step 2 to make a tip of ~ 10 $\mu$m. The needle was back-filled with Dent’s using a micropipette filler (250 $\mu$m ID, 350 $\mu$m OD, World Precision Instruments).  mk11g11 committed Sep 16, 2019 668 669  #### Experimental set-up  mk11g11 committed Feb 19, 2020 670 The microelectrode was inserted into a microelectrode holder containing a silver wire. The microelectrode was inserted into a headstage (HS-2A Asoclamp) and carefully lowered using a micromanipulator (Burleigh) into a recording chamber filled with Dent’s saline and resting on a stage of Axoscope 2 (Zeiss) microscope. The reference electrode was made with a glass capillary filled with 2 % agar in 3 M KCl. The reference electrode was placed in the recording chamber and connected to the amplifier headstage via a dish filled with 3 M KCl solution and a silver wire electrode. The cut head was placed in a recording chamber and a tight seal between the tip of the nose and the microelectrode was made by applying suction. The extracellular electrical signals from the pharynx were amplified by an Axoclamp-2B Microelectrode Amplifier, digitized by Digidata 1322A and recorded with Axoscope 9.2.  mk11g11 committed Sep 16, 2019 671 672 673 674 675 676 677 678  (ref:EPG-setup) **Experimental preparation for extracellular recordings from the *C. elegans* pharynx.** A diagram showing the set-up used for EPG experimentation. {r EPG-setup-method, fig.cap= "(ref:EPG-setup)", fig.scap = "Experimental preparation for extracellular recordings from the \\textit{C. elegans} pharynx.", fig.align='center', fig.pos = 'H', echo=FALSE} knitr::include_graphics("fig/methods/EPGsetup.png")  #### Experimental protocols  mk11g11 committed Feb 19, 2020 679 A cut head was placed in a recording chamber. The seal around the tip of the nose and the microelectrode was made and a worm was left for 5 minutes to acclimatise. Solutions changes were were achieved by gravity perfusion with a flow rate of ~ 1 mL/min. The pharyngeal pumping was recorded during control perfusion, drug perfusion and recovery into buffer in 3 equal 5-minute blocks giving a total time of the recording of 15 minutes.  mk11g11 committed Sep 16, 2019 680 681  #### Data acquisition and analysis  mk11g11 committed Feb 19, 2020 682 A single EPG reflects a contruction-relaxation of a pharyngeal muscle. It consists of a series of peaks, including e and E or excitatory peaks, I or inhibitory peak as well as r and R, or repolarising peaks (Figure \@ref(fig:example-epg-label)). The effects of exposure to drugs on three parameters were measured. (1) The pumping rate, which was derived by taking maximum pumping rate in a 10 second window. (2) The E/R ratio, which is the ratio between the ampliture of E and R spikes. This was measured by calculating the average of E/R ratios of all EPGs in the period of the maximum pumping. (2) The pump duration, which is the average duration of all EPGs in the period of the maximum pumping. If there were less then 10 EPGs, 10 consecutive peaks were taken to derive the final pump duration and E/R ratio value.  mk11g11 committed Sep 16, 2019 683 684 685 686  ### Microinjection to generate *C. elegans* transgenic lines ###{#microinjection} #### Preparation of a needle  mk11g11 committed Feb 19, 2020 687 Alluminosilicate capillaries SM100F-10 (1 mm external diameter, 0.5 mm internal diameter) needle was pulled with Narishige puller (model PC:10) using the following settings: step 1 at 99 $^\circ$C, step 2 at 79 $^\circ$C. The pulled needle was filled with 1 $\mu$L of injection mix and assembled into Transferman NK2 (Eppendorf) micromanipulator. Microinjection was performed with FemtoJet Microinjector (Eppendorf).  mk11g11 committed Sep 16, 2019 688 689 690 691  #### Generation of transgenic lines ##### Preparation of DNA microinjection mix  mk11g11 committed Feb 19, 2020 692 Test DNA plasmid was prepared with a Green Fluorescent Protein (GFP) co-injected marker to identify transgenic worms. Injection mix containing the the test plasmid at (5 ng/μL) and the co-injection marker (30 ng/μL) were resuspended in ddH~2~O and centrifuged at 15000 rpm for 5 minutes to precipitate aggregates. One $\mu$L of the cleared DNA mix was back filled into the injection needle.  mk11g11 committed Sep 16, 2019 693 694  ##### Injection  mk11g11 committed Feb 19, 2020 695 A single L4 + 1 was picked from NGM plate and immobilised by gently pressing it into a drop of Halocarbon oil 700 on 2 % agarose pad. The agarose pad was placed on a stage of Nikon Eclipse TE200 microscope and the syncytium of the anterior and/or posterior arm of the *C. elegans* gonad was injected with the DNA mix. Injected worms were gently liberated from the oil with a pick and placed on individual seeded NGM plates.  mk11g11 committed Sep 16, 2019 696   mk11g11 committed Oct 06, 2019 697 698 \newpage  mk11g11 committed Sep 16, 2019 699 ##### Screening of injected plates  mk11g11 committed Feb 19, 2020 700 The progeny of injected worms were viewed under the fluorescent microscope and GFP filter. Green F1 worms were picked individually onto separate seeded NGM plates and left to propagate. Plates containing green F2s were collected and kept as separate stable lines (Figure \@ref(fig:selection-process-label)).  mk11g11 committed Sep 16, 2019 701   mk11g11 committed Feb 19, 2020 702 (ref:selection-process) **Selection of transgenic worms.** L4 + 1 haermaphrodites are co-injected with selectivity marker (i.e. a vector containing gene encoding for GFP under the body wall muscle promoter). Green worms were selected and kept separately as separate lines.  mk11g11 committed Sep 16, 2019 703 704 705 706 707  {r selection-process-label, fig.cap="(ref:selection-process)", fig.scap = "Selection of transgenic worms.", fig.align='center', echo=FALSE, fig.pos = 'H'} knitr::include_graphics("fig/methods/select-transgenic-worms.png")   mk11g11 committed Oct 06, 2019 708 \newpage  mk11g11 committed Sep 16, 2019 709   mk11g11 committed Feb 19, 2020 710 ### Determination of human nAChR expression in the *C. elegans* pharynx by staining with conjugated $\alpha7$ selective antagonist FITC-$\alpha$-bungarotoxin(Bgtx) ###{#fitcmethod}  mk11g11 committed Sep 16, 2019 711 712  #### Worm preparation  mk11g11 committed Oct 06, 2019 713 Worms were submerged in 3 mL of Dent's saline in 5 cm Petri dish. To ease dissection, they were paralysed by placing the dish at -20 $^\circ$C for 5 minutes. Following this, the tip of the nose was cut perpendicular to the head to allow the cuticle to roll back and expose the pharynx. Next, the cut just below the terminal bulb was made and liberated pharynxes collected (Figure \@ref(fig:exposed-pharynx-label)).  mk11g11 committed Sep 16, 2019 714 715 716 717 718 719 720 721 722 723  (ref:exposed-pharynx-method) **Exposure of the *C. elegans* pharynx.** Using surgical blade, the cut was made at a tip of the nose and just below the terminal bulb (left image, black lines) to expose the pharynx (right). {r exposed-pharynx-label, fig.cap= "(ref:exposed-pharynx-method)", fig.scap = "Exposure of the \\textit{C. elegans} pharynx.", fig.align='center', echo=FALSE, fig.pos = 'H'} knitr::include_graphics("fig/methods/exposed-pharynx.png")  #### Staining  mk11g11 committed Feb 19, 2020 724 Exposed pharynxes were placed in 1 mL of Dent’s in a single well of a 12 well plate. FITC-$\alpha$Bgtx was added to the final concentration of 1 $\mu$g/mL. The plate was protected from light by covering in foil. The incubation proceeded for 1 hour at room temperature before being washed in 1 mL of Dent’s.  mk11g11 committed Sep 16, 2019 725 726  #### Imaging  mk11g11 committed Feb 19, 2020 727 $\alpha$Bgtx treated pharynxes were transferred onto 2 % agarose pad and covered with a slip. Images were taken immediately at 10x magnification. The preparation was exposed for 0.1 s and FITC filter was applied on NIKON E800 fluorescence microscope. Staining was quantified in ImageJ by subtracting background fluorescence from the fluorescence in the terminal bulb.