Team:Edinburgh/glgC

= glgC =

Amino acid sequence
MVSLEKNDHL MLARQLPLKS VALILAGGRG TRLKDLTNKR AKPA

VHFGGK FRIIDFALSN CINSGIRRMG VITQYQSHTL VQHIQRGWSF FNEEMNEFVD LL

PAQQRMKG ENWYRGTADA VTQNLDIIRR YKAEYVVILA GDHIYKQDYS RMLIDHVEKG

ARCTVACMPV PIEEASAFGV MAVDENDKII EFVEKPANPP SMPNDPSKSL ASMGIYVF

DA DYLYELLEED DRDENSSHDF GKDLIPKITE AGLAYAHPFP LSCVQSDPDA EPYWRD

VGTL EAYWKANLDL ASVVPELDMY DRNWPIRTYN ESLPPAKFVQ DRSGSHGMTL NSLV

SGGCVI SGSVVVQSVL FSRVRVNSFC NIDSAVLLPE VWVGRSCRLR RCVIDRACVI PE

GMVIGENA EEDARRFYRS EEGIVLVTRE MLRKLGHKQE R

Nucleotide sequence
3565981 atgccccaat aacatcagcc agaccgccgg ttttaagcag cgggaacatc tctgaacata

3566041 catgtaaaac ctgcattatc gctcctgttt atgccctaac ttccgtagca tttcgcgcgt

3566101 taccagcacg atgccttctt ctgaacgata gaaacgacgt gcatcttcct ctgcgttttc

3566161 accaatcacc atgccttccg gaataacaca agcacgatcg atgacgcagc ggcgcagacg

3566221 gcacgagcga cctacccata cttccggtaa caatacggcg gaatcaatgt tgcagaatga

3566281 attcacgcga acgcgcgaga acagaacgga ctgcaccacc accgaaccgg agatcacaca

3566341 accgccggaa accagtgagt taagggtcat cccgtggcta ccggagcgat cctgcacgaa

3566401 tttcgctggc ggtaatgatt cattgtaggt gcgaattggc caattgcgat cgtacatatc

3566461 cagttccggc accacagagg ccagatcgag gttcgctttc cagtaagctt ccagcgtacc

3566521 cacatcgcgc cagtacggct cggcatccgg gtcggattgt acgcaagaga gcgggaacgg

3566581 gtgcgcatag gccagaccgg cttcggtgat cttgggaatc aaatctttgc caaagtcgtg

3566641 gctggagttc tcatcgcgat cgtcttcttc cagcagttca tacagatagt cggcgtcaaa

3566701 gacgtagata cccatactcg ccagagattt gctcggatcg ttcggcattg acggcgggtt

3566761 agcaggtttt tcaacgaatt cgataatttt atcgttctca tcaaccgcca taacgccaaa

3566821 tgcggaggct tcttcaatcg gtactggcat acaagcaacg gtgcaacgtg cgcctttttc

3566881 gacgtgatcg ataagcatac gcgagtagtc ttgcttgtag atatggtcgc ccgccaggat

3566941 caccacgtat tccgctttat aacggcggat aatgtcgagg ttttgggtga ccgcatctgc

3567001 ggtgccgcga taccagtttt cccctttcat tctctgctgt gctggcagca gatcgacaaa

3567061 ctcgttcatt tcttcattga agaatgacca gccgcgctga atgtgctgca ccagagtgtg

3567121 ggactggtac tgggtgatca cgcccatacg acggatcccg gagttgatgc agttagacag

3567181 cgcaaagtcg ataatgcgga acttaccgcc gaagtgtacg gccggttttg ctcgcttatt

3567241 ggttaaatcc ttcaggcggg taccacgtcc tcccgccagt atcagggcaa cagatttcaa

3567301 tggcagctgg cgcgccaaca ttaagtgatc gttcttctct aaactaacca tgactaactc

3567361 cttttttatc atctctggaa cacacacaat ccgtgtgcag gtccctgcca gacagccgta

Complement

tacggctgtctggcagggacctgcacacggattgtgtgtgttccagagat 50

gataaaaaaggagttagtcatggttagtttagagaagaacgatcacttaa 100

tgttggcgcgccagctgccattgaaatctgttgccctgatactggcggga 150

ggacgtggtacccgcctgaaggatttaaccaataagcgagcaaaaccggc 200

cgtacacttcggcggtaagttccgcattatcgactttgcgctgtctaact 250

gcatcaactccgggatccgtcgtatgggcgtgatcacccagtaccagtcc 300

cacactctggtgcagcacattcagcgcggctggtcattcttcaatgaaga 350

aatgaacgagtttgtcgatctgctgccagcacagcagagaatgaaagggg 400

aaaactggtatcgcggcaccgcagatgcggtcacccaaaacctcgacatt 450

atccgccgttataaagcggaatacgtggtgatcctggcgggcgaccatat 500

ctacaagcaagactactcgcgtatgcttatcgatcacgtcgaaaaaggcg 550

cacgttgcaccgttgcttgtatgccagtaccgattgaagaagcctccgca 600

tttggcgttatggcggttgatgagaacgataaaattatcgaattcgttga 650

aaaacctgctaacccgccgtcaatgccgaacgatccgagcaaatctctgg 700

cgagtatgggtatctacgtctttgacgccgactatctgtatgaactgctg 750

gaagaagacgatcgcgatgagaactccagccacgactttggcaaagattt 800

gattcccaagatcaccgaagccggtctggcctatgcgcacccgttcccgc 850

tctcttgcgtacaatccgacccggatgccgagccgtactggcgcgatgtg 900

ggtacgctggaagcttactggaaagcgaacctcgatctggcctctgtggt 950

gccggaactggatatgtacgatcgcaattggccaattcgcacctacaatg 1000

aatcattaccgccagcgaaattcgtgcaggatcgctccggtagccacggg 1050

atgacccttaactcactggtttccggcggttgtgtgatctccggttcggt 1100

ggtggtgcagtccgttctgttctcgcgcgttcgcgtgaattcattctgca 1150

acattgattccgccgtattgttaccggaagtatgggtaggtcgctcgtgc 1200

cgtctgcgccgctgcgtcatcgatcgtgcttgtgttattccggaaggcat 1250

ggtgattggtgaaaacgcagaggaagatgcacgtcgtttctatcgttcag 1300

aagaaggcatcgtgctggtaacgcgcgaaatgctacggaagttagggcat 1350

aaacaggagcgataatgcaggttttacatgtatgttcagagatgttcccg 1400

ctgcttaaaaccggcggtctggctgatgttattggggcat 1440

Sequence map
tacggctgtctggcagggacctgcacacggattgtgtgtgttccagagat 50

atgccgacagaccgtccctggacgtgtgcctaacacacacaaggtctcta

1 2 3 4 5 6 7 8 9 10

gataaaaaaggagttagtc atg gtt agt tta gag aag aac gat cac tta a 100

ctattttttcctcaatcag tac caa tca aat ctc ttc ttg cta gtg aat t

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

tg ttg gcg cgc cag ctg cca ttg aaa tct gtt gcc ctg ata ctg gcg gga 150

ac aac cgc gcg gtc gac ggt aac ttt aga caa cgg gac tat gac cgc cct

28 29 KpnI 32 33 34 35 36 37 38 39 40 41 42 43 4

gga cgt ggt acc cgc ctg aag gat tta acc aat aag cga gca aaa ccg gc 200

cct gca cca tgg gcg gac ttc cta aat tgg tta ttc gct cgt ttt ggc cg

4 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

c gta cac ttc ggc ggt aag ttc cgc att atc gac ttt gcg ctg tct aac t 250

g cat gtg aag ccg cca ttc aag gcg taa tag ctg aaa cgc gac aga ttg a

61 62 63 64 BamHI 67 68 69 70 71 72 73 74 75 76 77

gc atc aac tcc ggg atc cgt cgt atg ggc gtg atc acc cag tac cag tcc 300

cg tag ttg agg ccc tag gca gca tac ccg cac tag tgg gtc atg gtc agg

78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 9

cac act ctg gtg cag cac att cag cgc ggc tgg tca ttc ttc aat gaa ga 350

gtg tga gac cac gtc gtg taa gtc gcg ccg acc agt aag aag tta ctt ct

4 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 1

a atg aac gag ttt gtc gat ctg ctg cca gca cag cag aga atg aaa ggg g 400

t tac ttg ctc aaa cag cta gac gac ggt cgt gtc gtc tct tac ttt ccc c

11 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127

aa aac tgg tat cgc ggc acc gca gat gcg gtc acc caa aac ctc gac att 450

tt ttg acc ata gcg ccg tgg cgt cta cgc cag tgg gtt ttg gag ctg taa

128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 14

atc cgc cgt tat aaa gcg gaa tac gtg gtg atc ctg gcg ggc gac cat at 500

tag gcg gca ata ttt cgc ctt atg cac cac tag gac cgc ccg ctg gta ta

4 145 146 147 148 149 150 151 152 153 ClaI 156 157 158 159 160 1

c tac aag caa gac tac tcg cgt atg ctt atc gat cac gtc gaa aaa ggc g 550

g atg ttc gtt ctg atg agc gca tac gaa tag cta gtg cag ctt ttt ccg c

61 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177

ca cgt tgc acc gtt gct tgt atg cca gta ccg att gaa gaa gcc tcc gca 600

gt gca acg tgg caa cga aca tac ggt cat ggc taa ctt ctt cgg agg cgt

178 179 180 181 182 183 184 185 186 187 188 189 190 EcoRI 193 19

ttt ggc gtt atg gcg gtt gat gag aac gat aaa att atc gaa ttc gtt ga 650

aaa ccg caa tac cgc caa cta ctc ttg cta ttt taa tag ctt aag caa ct

4 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 2

a aaa cct gct aac ccg ccg tca atg ccg aac gat ccg agc aaa tct ctg g 700

t ttt gga cga ttg ggc ggc agt tac ggc ttg cta ggc tcg ttt aga gac c

11 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227

cg agt atg ggt atc tac gtc ttt gac gcc gac tat ctg tat gaa ctg ctg 750

gc tca tac cca tag atg cag aaa ctg cgg ctg ata gac ata ctt gac gac

228 229 230 231 PvuI 234 235 236 237 238 239 241 241 242 243 24

gaa gaa gac gat cgc gat gag aac tcc agc cac gac ttt ggc aaa gat tt 800

ctt ctt ctg cta gcg cta ctc ttg agg tcg gtg ctg aaa ccg ttt cta aa

4 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 2

g att ccc aag atc acc gaa gcc ggt ctg gcc tat gcg cac ccg ttc ccg c 850

c taa ggg ttc tag tgg ctt cgg cca gac cgg ata cgc gtg ggc aag ggc g

61 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277

tc tct tgc gta caa tcc gac ccg gat gcc gag ccg tac tgg cgc gat gtg 900

ag aga acg cat gtt agg ctg ggc cta cgg ctc ggc atg acc gcg cta cac

278 279 280 HindIII 284 285 286 287 288 289 290 291 292 293 29

ggt acg ctg gaa gct tac tgg aaa gcg aac ctc gat ctg gcc tct gtg gt 950

cca tgc gac ctt cga atg acc ttt cgc ttg gag cta gac cgg aga cac ca

4 295 296 297 298 299 300 PvuI 304 305 306 307 308 309 310 3

g ccg gaa ctg gat atg tac gat cgc aat tgg cca att cgc acc tac aat g 1000

c ggc ctt gac cta tac atg cta gcg tta acc ggt taa gcg tgg atg tta c

11 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327

aa tca tta ccg cca gcg aaa ttc gtg cag gat cgc tcc ggt agc cac ggg 1050

tt agt aat ggc ggt cgc ttt aag cac gtc cta gcg agg cca tcg gtg ccc

328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 34

atg acc ctt aac tca ctg gtt tcc ggc ggt tgt gtg atc tcc ggt tcg gt 1100

tac tgg gaa ttg agt gac caa agg ccg cca aca cac tag agg cca agc ca

4 345 346 347 348 349 350 351 352 353 354 355 356 EcoRI 359 360 3

g gtg gtg cag tcc gtt ctg ttc tcg cgc gtt cgc gtg aat tca ttc tgc a 1150

c cac cac gtc agg caa gac aag agc gcg caa gcg cac tta agt aag acg t

61 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377

ac att gat tcc gcc gta ttg tta ccg gaa gta tgg gta ggt cgc tcg tgc 1200

tg taa cta agg cgg cat aac aat ggc ctt cat acc cat cca gcg agc acg

378 379 380 381 382 383 ClaIPvuI 387 388 389 390 391 392 393 39

cgt ctg cgc cgc tgc gtc atc gat cgt gct tgt gtt att ccg gaa ggc at 1250

gca gac gcg gcg acg cag tag cta gca cga aca caa taa ggc ctt ccg ta

4 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 4

g gtg att ggt gaa aac gca gag gaa gat gca cgt cgt ttc tat cgt tca g 1300

c cac taa cca ctt ttg cgt ctc ctt cta cgt gca gca aag ata gca agt c

11 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

aa gaa ggc atc gtg ctg gta acg cgc gaa atg cta cgg aag tta ggg cat 1350

tt ctt ccg tag cac gac cat tgc gcg ctt tac gat gcc ttc aat ccc gta

428 429 430 431 *

aaa cag gag cga taa tgcaggttttacatgtatgttcagagatgttcccg 1400

ttt gtc ctc gct att acgtccaaaatgtacatacaagtctctacaagggc

ctgcttaaaaccggcggtctggctgatgttattggggcat 1440

gacgaattttggccgccagaccgactacaataaccccgta

EcoRI FOUND 2.

PstI not found.

SpeI not found.

XbaI not found.

Two EcoRI sites are present at +570 and +1137.

Thus a digest with EcoRI and PstI should yield a vector fragment plus fragments of 570, 567, and 228 bp.

BioBrickTM primers
F primer: atg gtt agt tta gag aag aac gat c 25mer

R primer: tta tta tcg ctc ctg ttt atg ccc taa c

Mutagenesis
Site 1: codons 191 and 192, gaa-ttc, EF. Possible silent changes gag-ttc or gaa-ttt. We opted for gaa-ttt.

Forward: tgttgaaaaacctgctaacc - 54°C

Reverse: aattcgataattttatcgttc -52°C

Site 2: codons 356 to 358, gtg-aat-tca, VNS. Possible silent changes gtc-aat-tca, or gtg-aac-tca, no good possibility changing base 1 of the serine codon only. We went for g-aac-tca.

Forward: atcattctgcaacattgattcc - 54°C

Reverse: ttcacgcgaacgcgcg - 54°C

The mutation required to produce glgC16 is G336D, GGC changed to GAC.

Forward: acggttgtgtgatctccg - 54°C

Reverse: cggaaaccagtgagttaag - 56°C