Difference between revisions of "Hypo proj pract"

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(Created page with "=Introduction= Project 41, GFP-CFP expression (joh_j41) ==Goal== Produce a plasmid for the expression of the GFP-CFP (green and cyan fluorescent proteins) in bacteria to show...")
 
 
(10 intermediate revisions by the same user not shown)
Line 190: Line 190:
 
*Purification: dPNI & kit
 
*Purification: dPNI & kit
 
*Gel:
 
*Gel:
−
*Remark:
+
[[File:Gel_ex.jpg|400px|thumb|center|Gel electrophoresis]]
 +
*Remark: No GC enhancer or DMSO was used
 
*Concentrations:
 
*Concentrations:
 +
{| class="wikitable"
 +
|-
 +
! Name !! concentration (ng/ul)
 +
|-
 +
| joh_j41_V(d019B) || 72
 +
|-
 +
| joh_j41_I1(d019C)|| 237
 +
|-
 +
| joh_j41_I2(d019D) || 212
 +
|}
 +
*Remarks: Should let my gel migrate more to have better ladder resolution
 +
 +
=== Assembly ===
 +
*Method: Gibson ligation
 +
*Strain: XL1Blue
 +
*Purification Yields:
 +
{| class="wikitable"
 +
|-
 +
! Name !! concentration (ng/ul)
 +
|-
 +
| joh_j41_p1(p01A4) || 112
 +
|-
 +
| joh_j41_p2(d01A5)|| 97
 +
|-
 +
| joh_j41_p3(d01A6) || 105
 +
|}
 +
*Sequencing Results:
 +
{| class="wikitable"
 +
|-
 +
! Name !! T7 !! pET-RP !! Status
 +
|-
 +
| joh_j41_p1(p01A4) || Good 100% YFP coverage, 10% CFP coverage || Point Mutation in base 521 || Discarded
 +
|-
 +
| joh_j41_p2(d01A5)|| Good 100% YFP coverage, 10% CFP coverage || 100% CFP coverage || Selected
 +
|-
 +
| joh_j41_p3(d01A6) || Template... || Template... || Discarded
 +
|}
 +
*Remarks: Could leave dPNI for 15 more minutes to ensure complete template digestion
 +
== Protein Expression ==
 +
*Strain : BL21
 +
*Medium : LB
 +
*Volume : 1L
 +
*Induction : IPTG
 +
*O.D. at Induction : 0.9
 +
*Purification protocol : Standard His & Strep
 +
*Yields after His-tag (mg/ml):
 +
::*H1:0.98 , H2:1.78 , H3 : 0.48 , H4 : 0.11 , H5 : 0
 +
*Yields after Strep-tag (mg/ml):
 +
::*S1:0 , S2:0.78 , S3 : 0.38 , S4 : 0.08 , S5 : 0
 +
*Final Yield:
 +
::*Volume : 60ul
 +
::*Concentration : 323uM
 +
*Gel:
 +
[[File:Coom_ex.png|400px|thumb|center|Protein gel electrophoresis]]
 +
*Remarks: Could try sonicating more to completely destroy the pellet

Latest revision as of 21:43, 29 August 2016

Introduction

Project 41, GFP-CFP expression (joh_j41)

Goal

Produce a plasmid for the expression of the GFP-CFP (green and cyan fluorescent proteins) in bacteria to show FRET behavior.

Author

John Doe (joh)

Date

12.02.2111

Design

Plasmid

  • Name: joh_j41_p
  • Len: 6940bp
  • Expression frame: 1854bp

Vector

  • Construction based on pET-51b(+)
  • Template: mgr_j5_p31 (p01AB)
  • Name: joh_j41_dV (d019B)
  • Sequence:
      1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 
     61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 
    121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 
    181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 
    241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 
    301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 
    361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 
    421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat 
    481 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 
    541 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 
    601 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 
    661 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 
    721 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 
    781 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 
    841 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 
    901 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 
    961 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 
   1021 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 
   1081 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 
   1141 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 
   1201 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 
   1261 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 
   1321 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 
   1381 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 
   1441 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg 
   1501 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac 
   1561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 
   1621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 
   1681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 
   1741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 
   1801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 
   1861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 
   1921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 
   1981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 
   2041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 
   2101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 
   2161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 
   2221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 
   2281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 
   2341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 
   2401 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac 
   2461 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg 
   2521 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 
   2581 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 
   2641 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg 
   2701 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga 
   2761 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg 
   2821 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa 
   2881 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt 
   2941 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt 
   3001 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct 
   3061 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac 
   3121 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag 
   3181 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc 
   3241 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc 
   3301 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc 
   3361 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 
   3421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 
   3481 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt 
   3541 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg 
   3601 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc 
   3661 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat 
   3721 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt 
   3781 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc 
   3841 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc 
   3901 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag 
   3961 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt 
   4021 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat 
   4081 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag 
   4141 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac 
   4201 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg 
   4261 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg 
   4321 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt 
   4381 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc 
   4441 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact 
   4501 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg 
   4561 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga 
   4621 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc 
   4681 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt 
   4741 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg 
   4801 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc 
   4861 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 
   4921 aaataatttt gtttaacttt aagaaggaga tataccatgg caagctggag ccacccgcag 
   4981 ttcgaaaagg gtgcagatga cgacgacaag gtaccggatc cgacgtcggt cgacaagctt 
   5041 gcggccgcag gagctccggg cttctcctca atttccgctc atcaccacca tcatcaccat 
   5101 caccaccact aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt 
   5161 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggat
  • Len: 5218bp

Inserts

  • Insert 1:
Name: joh_j41_dI1(d019C)
Sequence:

ATGTCTCTTTCAAAGCATGGCATCACACAAGAAATGCCGACGAAATACCATATGAAAGGC AGTGTCAATGGCCATGAATTCGAGATCGAAGGTGTAGGAACTGGACACCCTTACGAAGGG ACACACATGGCCGAATTAGTGATCATAAAGCCTGCGGGAAAACCCCTTCCATTCTCCTTT GACATACTGTCAACAGTCATTCAATACGGAAACAGATGCTTCACTAAGTACCCTGCAGAC CTGCCTGACTATTTCAAGCAAGCATACCCAGGTGGAATGTCATATGAAAGGTCATTTGTG TATCAGGATGGAGGAATTGCTACAGCGAGCTGGAACGTTGGTCTCGAGGGAAATTGCTTC ATCCACAAATCCACCTATCTTGGTGTAAACTTTCCTGCTGATGGACCCGTAATGACAAAG AAGACAATTGGCTGGGATAAAGCCTTTGAAAAAATGACTGGGTTCAATGAGGTGTTAAGA GGTGATGTGACTGAGTTTCTTATGCTCGAAGGAGGTGGTTACCATTCATGCCAGTTTCAC TCCACTTACAAACCAGAGAAGCCGGTCGAACTGCCCCCGAATCATGTCATAGAACATCAC ATTGTGAGGACCGACCTTGGCAAGACTGCAAAAGGCTTCATGGTCAAGCTGGTACAACAT GCTGCGGCTCATGTTAACCCTTTGAAGGTTCAATAA

Len : 969bp
  • Insert 2:
Name: joh_j41_dI2(d019D)
Sequence:

ATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGT GATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACAAACGGA AAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTT GTCACTACTTTGACTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAG CATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATCTTTC AAAGATGACGGGACCTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTT AATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAA TTGGAATACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGA ATCAAAGCTAACTTCAAAATTAGACACAACGTTGAAGATGGAAGCGTTCAACTAGCAGAC CATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTAC CTGTCCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTT CTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAAGGCCT GCAGCAAACGACGAAAACTACGCTGCATCAGTT

Len : 753bp

Primers

Name Sequence Melting Temperature (°C)
joh_j41_a1(a019E) ACCGGCATATTTTATCGGTACTGGCTTTCCATTCGAC 67
joh_j41_a2(a019F) AGTACCGATAAAATATGCCGGTGTCAGTTCGGTAC 67
joh_j41_a3(a01A0) TGTTGAAAATGAAGGATCCGAAATCGGTACTGGCTTTC 68
joh_j41_a4(a01A1) TTCGGATCCTTCATTTTCAACAACATCAACGCTTGCAAC 67
joh_j41_a5(a01A2) TGGTAGTGAAGCAAGCGCCAGCATTATTG 66
joh_j41_a6(a01A3) AATGCTGGCGCTTGCTTCACTACCACCGCTGCCG 66

Summary

final plasmid design

Protein

  • Construct name:joh_j41_c (c01A4)
  • Sequence (coding frame only):

MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATNGKLTLKFICTT GKLPVPWPTLVTTFTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISF KDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNV YITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHY LSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKRPAANDENYAAS VMFKGIVEGIGIIEKIDIYTDLDKYAIRFPENMLNGIKKESSIMFNGCFLT VTSVNSNIVWFDIFEKEARKLDTFREYKVGDRVNLGTFPKFGAASGGHIL SARISCVASIIEIIENEDYQQMWIQIPENFTEFLIDKDYIAVDGISLTID TIKNNQFFISLPLKIAQNTNMKWRKKGDKVNVELSNKINANQCWGSGGAT HHHHHHH

  • Len: 458 AA
  • Molecular Weight: 51734.61 Da
  • Extinction coefficient: 54110

Experimental

Plasmid

Segments

  • Polymerase used: Open Taq
  • Annealing temp: 61°C
  • Purification: dPNI & kit
  • Gel:
Gel electrophoresis
  • Remark: No GC enhancer or DMSO was used
  • Concentrations:
Name concentration (ng/ul)
joh_j41_V(d019B) 72
joh_j41_I1(d019C) 237
joh_j41_I2(d019D) 212
  • Remarks: Should let my gel migrate more to have better ladder resolution

Assembly

  • Method: Gibson ligation
  • Strain: XL1Blue
  • Purification Yields:
Name concentration (ng/ul)
joh_j41_p1(p01A4) 112
joh_j41_p2(d01A5) 97
joh_j41_p3(d01A6) 105
  • Sequencing Results:
Name T7 pET-RP Status
joh_j41_p1(p01A4) Good 100% YFP coverage, 10% CFP coverage Point Mutation in base 521 Discarded
joh_j41_p2(d01A5) Good 100% YFP coverage, 10% CFP coverage 100% CFP coverage Selected
joh_j41_p3(d01A6) Template... Template... Discarded
  • Remarks: Could leave dPNI for 15 more minutes to ensure complete template digestion

Protein Expression

  • Strain : BL21
  • Medium : LB
  • Volume : 1L
  • Induction : IPTG
  • O.D. at Induction : 0.9
  • Purification protocol : Standard His & Strep
  • Yields after His-tag (mg/ml):
  • H1:0.98 , H2:1.78 , H3 : 0.48 , H4 : 0.11 , H5 : 0
  • Yields after Strep-tag (mg/ml):
  • S1:0 , S2:0.78 , S3 : 0.38 , S4 : 0.08 , S5 : 0
  • Final Yield:
  • Volume : 60ul
  • Concentration : 323uM
  • Gel:
Protein gel electrophoresis
  • Remarks: Could try sonicating more to completely destroy the pellet
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