20.109(S07): Agarose gel electrophoresis: Difference between revisions

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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 18:36, 15 December 2006


20.109: Laboratory Fundamentals of Biological Engineering

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Genome Engineering        Biophysical Signal Measurement        Expression Engineering        Biomaterial Engineering       

Introduction

Protocols

Part 1: Cloning

  1. anneal oligo
  2. run oligos and bkb on gel
  3. excise frags and purify from gel

Part 2: Infecting cells with phage

  1. 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
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
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

Reagents list

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