Difference between revisions of "20.109(S07): Agarose gel electrophoresis"
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DONE! | DONE! | ||
==For next time== | ==For next time== | ||
+ | ==Coding sequences/replication sequences== | ||
+ | |||
+ | {| border="1" | ||
+ | ! genetic element | ||
+ | ! bp in M13K07 [[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/sequences/m13ko7.txt]] | ||
+ | ! BBa_ | ||
+ | ! notes on standardization | ||
+ | |- | ||
+ | | gene II CDS | ||
+ | | 8268- 831 | ||
+ | | BBa_M13002 | ||
+ | | will need to disable internal RBS that allows gX transcription from within gII coding sequence | ||
+ | |- | ||
+ | | gene X CDS | ||
+ | | 496- 831 | ||
+ | | BBa_M13010 | ||
+ | | need to unstuff from inside gII | ||
+ | |- | ||
+ | | gene V CDS | ||
+ | | 843-1106 | ||
+ | | BBa_M13005 | ||
+ | | RBS overlaps stop of upstream gII, gX | ||
+ | |- | ||
+ | | gene VII CDS | ||
+ | | 1108-1209 | ||
+ | | BBa_M13007 | ||
+ | | stop codon overlaps with start of dwstm gIX | ||
+ | |- | ||
+ | | gene IX CDS | ||
+ | | 1206-1304 | ||
+ | | BBa_M13009 | ||
+ | | start codon overlaps with stop of upstm gVII <br> stop codon overlaps with start of dwstm gVIII | ||
+ | |- | ||
+ | | gene VIII CDS | ||
+ | | 1301-1522 | ||
+ | | BBa_M13008 | ||
+ | | start codon overlaps with stop of upstm gIX <br> might want different BBa with codon varied ORF <br> might want to include silent cloning sites for N-terminal fusions after first Ala of mature protein | ||
+ | |- | ||
+ | | gene III CDS | ||
+ | | 1579-2853 | ||
+ | | BBa_M13003 | ||
+ | | start codon is GTG <br> RBS is within transcriptional terminator for upstream gVIII <br> unique BamHI site in domain 2 may be useful for cloning | ||
+ | |- | ||
+ | | gene VI CDS | ||
+ | | 2856-3194 | ||
+ | | BBa_M13006 | ||
+ | | | ||
+ | |- | ||
+ | | gene I CDS | ||
+ | | 3196-4242 | ||
+ | | BBa_M13001 | ||
+ | | will need to disable internal RBS that leads to gXI transcription <br> | ||
+ | weach rho-dependent terminator limits gI txn <br> rare codons limit gI tln | ||
+ | |- | ||
+ | | gene XI CDS | ||
+ | | 3916-4242 | ||
+ | | BBa_M13011 | ||
+ | | need to unstuff from inside gI | ||
+ | |- | ||
+ | | gene IV CDS | ||
+ | | 4220-5500 | ||
+ | | BBa_M13004 | ||
+ | | start codon and RBS are within gI/gXI coding sequence<br> stop codon is within M13 ori on M13K07 | ||
+ | |- | ||
+ | | M13 origin of replication (portion) | ||
+ | | 5488-5830 | ||
+ | | | ||
+ | | | ||
+ | |- | ||
+ | | p15A origin of replication (counterclockwise) | ||
+ | | 6591-6046 | ||
+ | | | ||
+ | | | ||
+ | |- | ||
+ | | Tn903 kanamycin resistance CDS | ||
+ | | 7956-7141 | ||
+ | | | ||
+ | | | ||
+ | |- | ||
+ | | M13 origin of replication (portion) | ||
+ | | 8093-8130 | ||
+ | | | ||
+ | | | ||
+ | |} | ||
+ | |||
+ | ==RBS as identified in Gene (1980) 11:129-148== | ||
+ | Renumbered bp according to M13K07 genome annotation <br> | ||
+ | 16S RNA 3'OHAUUCCUCCACUAG-------- <br> | ||
+ | Note: All RBS are directly followed by ATG of coding seq then a codon starting with A, except gene 6, gene 7 and gene I which follow ATG with G (genes 1 and 7) or C (gene 6). Genes 1, 6 and 7 are translated only to low levels in vivo and in vitro. | ||
+ | |||
+ | {| border="1" | ||
+ | ! genetic element | ||
+ | ! bp in M13K07 [[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/sequences/m13ko7.txt]] | ||
+ | ! BBa_ | ||
+ | ! seq | ||
+ | |- | ||
+ | | gene II RBS | ||
+ | | 8252-8267 | ||
+ | | BBa_M13502 | ||
+ | | ATCAACCGGGGTACAT | ||
+ | |- | ||
+ | | gene X RBS | ||
+ | | 480-495 | ||
+ | | BBa_M13510 | ||
+ | | ATTTGAGGGGGATTCA | ||
+ | |- | ||
+ | | gene V RBS | ||
+ | | 827-842 | ||
+ | | BBa_M13505 | ||
+ | | CATAAGGTAATTCACA | ||
+ | |- | ||
+ | | gene VII RBS | ||
+ | | 1092-1107 | ||
+ | | BBa_M13507 | ||
+ | | GTTCCGGCTAAGTAAC | ||
+ | |- | ||
+ | | gene IX RBS | ||
+ | | 1190-1205 | ||
+ | | BBa_M13509 | ||
+ | | TCGCTGGGGGTCAAAG | ||
+ | |- | ||
+ | | gene VIII RBS | ||
+ | | 1285-1300 | ||
+ | | BBa_M13508 | ||
+ | | TAATGGAAACTTCCTC | ||
+ | |- | ||
+ | | gene III RBS | ||
+ | | 1563-1578 | ||
+ | | BBa_M13503 | ||
+ | | TTTGGAGA TTTTCAAC | ||
+ | |- | ||
+ | | gene VI RBS | ||
+ | | 2840-2855 | ||
+ | | BBa_M13506 | ||
+ | | ATAAGGAGTCTTAATC | ||
+ | |- | ||
+ | | gene I RBS | ||
+ | | 3180-3195 | ||
+ | | BBa_M13501 | ||
+ | | GATTGGGATAAATAAT | ||
+ | |- | ||
+ | | gene XI RBS | ||
+ | | 3900-3915 | ||
+ | | BBa_M13511 | ||
+ | | AATTTAGGTCAGAAG <br> RBS not identified in Gene 1980 paper | ||
+ | |- | ||
+ | | gene IV RBS | ||
+ | | 4204-4219 | ||
+ | | BBa_M13504 | ||
+ | | AAAAAAGGTAATTCAA | ||
+ | |} | ||
+ | |||
+ | ==Promoters as identified in Gene (1980) 11:129-148== | ||
+ | Renumbered bp according to M13K07 genome annotation <br> | ||
+ | -10 is TATAATpu centered around -8 from mRNA initiation point <br> | ||
+ | -35 is TGTTGACAATT centered around -35 from mRNA initiation point <br> | ||
+ | +2 position is often T<br> | ||
+ | Many of these promoters were identified as DNA fragments that could bind RNAP | ||
+ | |||
+ | {| border="1" | ||
+ | ! genetic element | ||
+ | ! bp in M13K07 <br> [[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/sequences/m13ko7.txt]] | ||
+ | ! BBa_ | ||
+ | ! seq <br>matches to -35 and -10 concensus in bold<br> positions -30, -20, -10 and zero relative to mRNA start in <font color = red> red </font color><br> differences with fd in <font color= purple> purple </font color> | ||
+ | |- | ||
+ | | gene II promoter | ||
+ | | 8188-8235 | ||
+ | | BBa_M13102 | ||
+ | | TA<b>T</b>TAAC<b>GTT</b>T<b>AC<font color = red>A</font color>ATTT</b>AAATA<font color= red>T</font color><b>T</b>TGC<b>T</b>TA<b>TA<font color = red>C</font color>AAT</b>C<font color = purple>T</font color>TCCT<font color = red>G</font color>T<b>T</b>TT | ||
+ | |- | ||
+ | | gene X promoter | ||
+ | | 381-428 | ||
+ | | BBa_M13110 | ||
+ | | TC<b>T</b>TTT<b>TG</b>A<b>T G</b>CA<font color =red><b>A</b></font color>T<font color = purple>C</font color>CGC<b>T</b>TTG<font color = red>C</font color><b>T</b>TCTGA C<b>TA<font color = red>T</font color>AAT</b>AG<font color = purple>T</font color> CAG<font color = red>G</font color>GTAA | ||
+ | |- | ||
+ | | gene V promoter | ||
+ | | 786-835 | ||
+ | | BBa_M13105 | ||
+ | | CCAACG<b>T</b>CC<b>TGAC</b>TG<font color = red>G</font color><b>T</b>ATAATGAG<font color = red>C</font color>AGT<b>T</b>C<b>TTA</b><font color = red>A</font color><b>AAT</b>CGCATA<font color = red>A</font color>GGTA | ||
+ | |- | ||
+ | | gene VII promoter | ||
+ | | | ||
+ | | | ||
+ | | driven from II, X, V promoters (BBa_M13102,_M13110,_M13105)? | ||
+ | |- | ||
+ | | gene IX promoter | ||
+ | | | ||
+ | | | ||
+ | | driven from II, X, V promoters (BBa_M13102,_M13110,_M13105)? | ||
+ | |- | ||
+ | | gene VIII promoter | ||
+ | | 1155-1201 | ||
+ | | BBa_M13108 | ||
+ | | AA<b>T</b>CTC C<b>GTTG</b>TA<font color=red>C</font color>T<b>T T</b>GT<b>TT</b>C<font color = red>G</font color>CGC T<b>TGGTA<font color = red>T</font color>AAT</b>CGCTGG<font color= red>G</font color>GGT C | ||
+ | |- | ||
+ | | gene III promoter | ||
+ | | 1500-1547 | ||
+ | | BBa_M13103 | ||
+ | | AA<b>T</b>TCACC<b>T</b>C<b>GA</b>A<font color = red><b>A</b></font color>GCAAGCTGA<font color = red>T</font color>AAACC<b>G</b>A<b>TA</b><font color = red>C</font color><b>AAT</b>TAAAGG<font color = red>C</font color>TCCT | ||
+ | |- | ||
+ | | gene VI | ||
+ | | 2716-2764 | ||
+ | | BBa_M13106 | ||
+ | | GG<b>T</b>AAACCA<b>T</b>ATG<b>A<font color = red>A</font color>TTT</b>TCTATT<font color = red>G</font color>AT<b>T</b>G<b>TG</b>AC<b>A</b><font color = red>A</font color><b>AAT</b>AAACTT<font color = red>A</font color>T<b>T</b>CC | ||
+ | |- | ||
+ | | gene I promoter | ||
+ | | 3086-3132 | ||
+ | | BBa_M13101 | ||
+ | | CCCGTC<b>T</b>AAT<b>G</b>CG<font color = red>C</font color>T <b>T</b>CCC<b>T</b>GT<font color = red>T</font color><b>T</b>T<b>T</b>A<b>TGTTA<font color = red>T</font color>T</b>C<b>T</b>CTCTGT<font color = red>A</font color>AAGG | ||
+ | |- | ||
+ | | gene XI promoter | ||
+ | | | ||
+ | | | ||
+ | | protein from translation re-initiation event within gene I transcript | ||
+ | |- | ||
+ | | gene IV promoter | ||
+ | | 4055-4103 | ||
+ | | BBa_M13104 | ||
+ | | TTGATAAA<b>TT</b>C<b>AC</b>T<b><font color = red>A</font color>TT</b>GACTCTT<font color = red>C</font color><b>T</b>CAGC<b>GT</b>CT<b><font color = red>T</font color>AAT</b>CTAAG<b>C</b><font color = red>T</font color>A<b>T</b>CG | ||
+ | |} | ||
+ | |||
+ | ==Terminator sequences== | ||
+ | Intergenic region I between genes VI and II, contains ori and PS <br> | ||
+ | Intergenic region II between genes VIII and III, contains transcription terminator | ||
+ | |||
==Reagents list== | ==Reagents list== |
Revision as of 01:36, 16 December 2006
Contents
Introduction
Protocols
Part 1: Cloning
- anneal oligo
- run oligos and bkb on gel
- excise frags and purify from gel
Part 2: Infecting cells with phage
- overnights of phage infected cells
DONE!
For next time
Coding sequences/replication sequences
genetic element | bp in M13K07 [[1]] | BBa_ | notes on standardization |
---|---|---|---|
gene II CDS | 8268- 831 | BBa_M13002 | will need to disable internal RBS that allows gX transcription from within gII coding sequence |
gene X CDS | 496- 831 | BBa_M13010 | need to unstuff from inside gII |
gene V CDS | 843-1106 | BBa_M13005 | RBS overlaps stop of upstream gII, gX |
gene VII CDS | 1108-1209 | BBa_M13007 | stop codon overlaps with start of dwstm gIX |
gene IX CDS | 1206-1304 | BBa_M13009 | start codon overlaps with stop of upstm gVII stop codon overlaps with start of dwstm gVIII |
gene VIII CDS | 1301-1522 | BBa_M13008 | start codon overlaps with stop of upstm gIX might want different BBa with codon varied ORF might want to include silent cloning sites for N-terminal fusions after first Ala of mature protein |
gene III CDS | 1579-2853 | BBa_M13003 | start codon is GTG RBS is within transcriptional terminator for upstream gVIII unique BamHI site in domain 2 may be useful for cloning |
gene VI CDS | 2856-3194 | BBa_M13006 | |
gene I CDS | 3196-4242 | BBa_M13001 | will need to disable internal RBS that leads to gXI transcription weach rho-dependent terminator limits gI txn |
gene XI CDS | 3916-4242 | BBa_M13011 | need to unstuff from inside gI |
gene IV CDS | 4220-5500 | BBa_M13004 | start codon and RBS are within gI/gXI coding sequence stop codon is within M13 ori on M13K07 |
M13 origin of replication (portion) | 5488-5830 | ||
p15A origin of replication (counterclockwise) | 6591-6046 | ||
Tn903 kanamycin resistance CDS | 7956-7141 | ||
M13 origin of replication (portion) | 8093-8130 |
RBS as identified in Gene (1980) 11:129-148
Renumbered bp according to M13K07 genome annotation
16S RNA 3'OHAUUCCUCCACUAG--------
Note: All RBS are directly followed by ATG of coding seq then a codon starting with A, except gene 6, gene 7 and gene I which follow ATG with G (genes 1 and 7) or C (gene 6). Genes 1, 6 and 7 are translated only to low levels in vivo and in vitro.
genetic element | bp in M13K07 [[2]] | BBa_ | seq |
---|---|---|---|
gene II RBS | 8252-8267 | BBa_M13502 | ATCAACCGGGGTACAT |
gene X RBS | 480-495 | BBa_M13510 | ATTTGAGGGGGATTCA |
gene V RBS | 827-842 | BBa_M13505 | CATAAGGTAATTCACA |
gene VII RBS | 1092-1107 | BBa_M13507 | GTTCCGGCTAAGTAAC |
gene IX RBS | 1190-1205 | BBa_M13509 | TCGCTGGGGGTCAAAG |
gene VIII RBS | 1285-1300 | BBa_M13508 | TAATGGAAACTTCCTC |
gene III RBS | 1563-1578 | BBa_M13503 | TTTGGAGA TTTTCAAC |
gene VI RBS | 2840-2855 | BBa_M13506 | ATAAGGAGTCTTAATC |
gene I RBS | 3180-3195 | BBa_M13501 | GATTGGGATAAATAAT |
gene XI RBS | 3900-3915 | BBa_M13511 | AATTTAGGTCAGAAG RBS not identified in Gene 1980 paper |
gene IV RBS | 4204-4219 | BBa_M13504 | AAAAAAGGTAATTCAA |
Promoters as identified in Gene (1980) 11:129-148
Renumbered bp according to M13K07 genome annotation
-10 is TATAATpu centered around -8 from mRNA initiation point
-35 is TGTTGACAATT centered around -35 from mRNA initiation point
+2 position is often T
Many of these promoters were identified as DNA fragments that could bind RNAP
genetic element | bp in M13K07 [[3]] |
BBa_ | seq matches to -35 and -10 concensus in bold positions -30, -20, -10 and zero relative to mRNA start in red differences with fd in purple |
---|---|---|---|
gene II promoter | 8188-8235 | BBa_M13102 | TATTAACGTTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTTT |
gene X promoter | 381-428 | BBa_M13110 | TCTTTTTGAT GCAATCCGCTTTGCTTCTGA CTATAATAGT CAGGGTAA |
gene V promoter | 786-835 | BBa_M13105 | CCAACGTCCTGACTGGTATAATGAGCAGTTCTTAAAATCGCATAAGGTA |
gene VII promoter | driven from II, X, V promoters (BBa_M13102,_M13110,_M13105)? | ||
gene IX promoter | driven from II, X, V promoters (BBa_M13102,_M13110,_M13105)? | ||
gene VIII promoter | 1155-1201 | BBa_M13108 | AATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAATCGCTGGGGGT C |
gene III promoter | 1500-1547 | BBa_M13103 | AATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCT |
gene VI | 2716-2764 | BBa_M13106 | GGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCC |
gene I promoter | 3086-3132 | BBa_M13101 | CCCGTCTAATGCGCT TCCCTGTTTTTATGTTATTCTCTCTGTAAAGG |
gene XI promoter | protein from translation re-initiation event within gene I transcript | ||
gene IV promoter | 4055-4103 | BBa_M13104 | TTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAATCTAAGCTATCG |
Terminator sequences
Intergenic region I between genes VI and II, contains ori and PS
Intergenic region II between genes VIII and III, contains transcription terminator