Library construction 1

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• Library construction 1
• extract mRNA
• divide by size
• screen subdivided mRNA for desired activity
  (e.g. in oocytes)
• synthesise first strand cDNA (TTTTTT primer)
• synthesise second strand DNA

2

• Library construction 2
• ligate into expression vector (here pcDNA3)

3
Transform bugs (nonpathogenic strain of E. coli)
Plasmid DNA

• Add growth medium
• Incubate 1 hr to express antibiotic resistance

Heat shock 42 C 1 min
Spread on agar with antibiotic, leave overnight
Competent E. coli
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After transformation (next morning)

• Divide agar plates into groups of colonies
• Library divided into 50-100 groups
• Each group contains 10000-20000 clones
• Extract DNA from each group
• Re-combine into 10-50 larger groups for
  screening

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250 mM CaCl2 1 mg plasmid DNA
140 mM NaCl 1.5 mM Na2HPO4 50 mM HEPES pH 7.05
Transfect cells with extracted DNA (use a cell
line that has few endogenous channels, e.g.
HEK293, CHO
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• Screen with Ca2 imaging
• For the cold/menthol receptor they used 1 mM
  menthol
• These images from an earlier study where the same
  group cloned the capsaicin receptor VR-1 using 3
  mM capsaicin

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After finding a positive pool

• Subdivide it as before
• This part is obviously much quicker if you used a
  large number of small pools and re-combined them

• Finally, the pool is subdivided down to a single
  clone
• This can now be sequenced

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Sequence and topology of TRPM8
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• How to predict the transmembrane regions?
• We have a 6TM channel with 8 potential TM domains!

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Transmembrane regions predicted by McKemy et al
S1 S2 S3 S4 S5 S6
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Can be predicted from experimentally determined
topology of a related channel - TRPC3
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Transmembrane regions predicted for TRPC3
S1 S2 S3 S4 S5 S6
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Unknown nucleotide sequence
gtunknown sequence ACTTCCGTAGAAGCTTCTGGAAAGAGGACAGA
AGCAGCAGGGAGGACTTG GATGTGGAACTCCATGATGCATCTCTCACCA
CCCGGCACCCCCTGCAGGC TCTTTTCATCTGGGCCATTCTTCAGAACAA
GAAGGAACTCTCCAAGGTCA TCTGGGAGCAAACCAAAGGCTGTACTCTG
GCCGCCTTGGGGGCCAGCAAA CTTCTGAAGACCCTGGCCAAAGTTAAGA
ATGATATCAACGCAGCTGGGGA ATCTGAGGAACTGGCTAATGAGTATGA
GAC
Six-frame translation ACTTCCGTAGAAGCTT sequence T.
.S..V..E..A.. frame 1 .L..P....K..L.. frame
2 ..F..R..R..S.. frame 3 ACTTCCGTAGAAGCTT sequen
ce TGAAGGCATCTTCGAA complement AAGCTTCTACGGAAGT r
everse complement K..L..L..R..K.. frame
-1 .S..F..Y..G..S.. frame -2 ..A..S..T..E.. frame
-3
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Six-frame translations of our unknown sequence
gtORF 2 to 7 Frame 2 2 aa LP gtORF 279 to
268 Frame -2 4 aa SHTH gtORF 264 to 238
Frame -2 9 aa PVPQIPQLR gtORF 234 to 226
Frame -2 3 aa YHS gtORF 11 to 64 Frame 2
18 aa KLLERGQKQQGGLGCGTP gtORF 222 to 151
Frame -2 24 aa LWPGSSEVCWPPRRPEYSLWFAPR gtORF
280 to 125 Frame -1 52 aa VSYSLASSSDSPAALI
SFLTLARVFRSLLAPKAARVQPLVCSQMTLESSFLF gtORF 147 to
109 Frame -2 13 aa PWRVPSCSEEWPR gtORF 105
to 79 Frame -2 9 aa KEPAGGAGW gtORF 1 to
204 Frame 1 68 aa TSVEASGKRTEAAGRTWMWNSMMHL
SPPGTPCRLFSSGPFFRTRRNSPRSSGSKPKAVLWPPWGPANF gtORF
68 to 223 Frame 2 52 aa CISHHPAPPAGSFHLGHSS
EQEGTLQGHLGANQRLYSGRLGGQQTSEDPGQS gtORF 227 to
229 Frame 2 1 aa E gtORF 233 to 253 Frame
2 7 aa YQRSWGI gtORF 257 to 265 Frame 2
3 aa GTG gtORF 272 to 274 Frame 2 1
aa V gtORF 3 to 278 Frame 3 92
aa FRRSFWKEDRSSREDLDVELHDASLTTRHPLQALFIWAILQNKKELS
KVIWEQTKGCTLAALGASKLLKTLAKVKNDINAAGESEELANEYE gtORF
208 to 279 Frame 1 24 aa RPWPKLRMISTQLGNLR
NWLMSMR gtORF 278 to 280 Frame 2 1
aa D gtORF 75 to 1 Frame -2 25
aa EMHHGVPHPSPPCCFCPLSRSFYGS
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Useful six-frame translations
gtORF 280 to 125 Frame -1 52
aa VSYSLASSSDSPAALISFLTLARVFRSLLAPKAARVQPLVCSQMTLE
SSFLF gtORF 1 to 204 Frame 1 68
aa TSVEASGKRTEAAGRTWMWNSMMHLSPPGTPCRLFSSGPFFRTRRNS
PRSSGSKPKAVLWPPWGPANF gtORF 68 to 223 Frame 2
52 aa CISHHPAPPAGSFHLGHSSEQEGTLQGHLGANQRLYSGRLGG
QQTSEDPGQS gtORF 3 to 278 Frame 3 92
aa FRRSFWKEDRSSREDLDVELHDASLTTRHPLQALFIWAILQNKKELS
KVIWEQTKGCTLAALGASKLLKTLAKVKNDINAAGESEELANEYE
We will use these to do a BLAST search BLAST
Basic Local Alignment Search Tool
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