Team:NYMU-Taipei/Project/Time Regulation/Reloxilator

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== Experimental Results ==
== Experimental Results ==
[[Image:Reloxilator_construct_schedule.png|695px]]
[[Image:Reloxilator_construct_schedule.png|695px]]
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This is a picture of all the constructs, and green constructs are the ones that were successfully created.
== References ==
== References ==

Latest revision as of 01:30, 30 October 2008

NYMU Banner-wider 965px.png

Our Reloxilator (Relaxation Oscillator): A four-part system consisting of an oscillator, a synchronizer, a tuner, and a reporter.

NYMU reloxilator.png

Contents

Brief Description

The oscillator is a two-component [http://en.wikipedia.org/wiki/Relaxation_Oscillator relaxation oscillator]. The first component consists of the PRE promoter and the gene CII in a positive feedback loop, and both originating from from the λ phage. The second component uses the HtlB gene from E. coli attached to the same promoter as the first component. The HtlB gene produces HtlB proteins that degrade CII proteins.

The synchronizer allows near-full synchronization between different cells after about two time periods of oscillation1. The main parts of the synchronizer is made up of the PLux promoter, and the LuxI and LuxR genes, all coming from the Vibrio Fischeri organism. PRE and the CII gene are the same as the parts used in the oscillator, tying the oscillator and synchronizer together.

The Tuner is a way to control the time period of the system by inhibiting the rate of degradation HtlB has on CII in the oscillator. The protein used is the CIIICd, a DNA synthesised version of the CIIIC protein from the study done by Halder et al2.

The Reporter is the promoter PRE (the same part used in the oscillator) attached to a green flurorescent protein gene. It is used in various tests to check many of the components in the system.


System Description

This section houses the description of our system and the we use.

The Oscillator

A two-component oscillator consisting of two genes: one that induces both genes, while the second gene represses the first one. This oscillation is much like the oscillation of a [http://en.wikipedia.org/wiki/Relaxation_Oscillator capacitor charging and discharging].

Components

Related Biobricks

  • [http://partsregistry.org/Part:BBa_R0053 BBa_R0053] works but comes from a different phage (P22). Therefore using it means no proof of oscillation with HtlB [citation required]. Just realised it doesn't mean we can't try it.
    • [http://partsregistry.org/Part:BBa_I7108 BBa_I7108] uses BBa_R0053 and produces little GFP output. But that may be good for us?
    • [http://partsregistry.org/Part:BBa_R1053 BBa_R1053] is a standardised version of it.
    • [http://partsregistry.org/Part:BBa_C0053 BBa_C0053], the related coding region that activates BBa_R0053.

Problems

  • [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=16131068 HtlB] already exists in the strain of E. coli we're going to use (Escherichia coli K12 strain MG1655 (tax-id:511145).
    1. Go ahead with the experiment, and then find out whether the amount that already exists would significantly impact our results or not by checking with a prediction.
  • Due to HtlB already existing in E. coli, if the initial concentration of CII isn't enough, would the oscillator never start?

References

  • McMillen, D. et al (2002) "[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=117365 Synchronizing genetic relaxation oscillators by intercell signalling]", Proc Natl Acad Sci USA, Vol. 22, No. 3, pp. 679-684.
    • Where the oscillator idea came from.


The Synchronizer

The synchronizer integrates the use of cell quorum sensing to introduce synchronization of intercell oscillations.

Components


Component positions on Vibrio Fischeri

We performed a bunch of sequence alignments between the sequences from the Ecocyc database and the Biobrick parts to determine position of those components on Vibrio Fischeri. Orange colour means transcription/translation in reverse direction.
norm means normalised (norm=x-1050153+1)

Component length Start End norm start norm end Source
LuxI 573 1050153 1050725 1 573 [http://biocyc.org/VFIS312309/NEW-IMAGE?type=GENE&object=VFA0924 Biocyc]
BBa_C0161 585 1050150 1050734 -2 582 [http://partsregistry.org/Part:BBa_C0161 BBa_c0161] and sequence alignment[2] (87.3% consequence.)
BBa_R0062 (pLUX-right) 55 1050749 1050803 597 651 [http://partsregistry.org/Part:BBa_R0062:Design BBa_R0062] and sequence alignment[2] (72.7% consequence).
55->57 1050784 1050840 632 688 sequence alignment[1] (56% consensus and added 2bp). Looks wrong.
BBa_R0061 30->28 1050781 1050808 627 656 [http://partsregistry.org/wiki/index.php/Part:BBa_R0061 BBa_R0061] and sequence alignment[1] (75% consensus, but they deleted the first 2bp).
30->43 1050775 1050817 623 665 sequence alignment[2] (76.5% consensus. This looks wrong.)
BBa_R0063 (pLUX-left) 151->150 1050784 1050933 632 781 [http://partsregistry.org/Part:BBa_R0063:Design BBa_R0063] and sequence alignment[1] (64.5% consensus and removed 1bp).
BBa_c0062 756->753 1050941 1051693 789 1541 [http://partsregistry.org/Part:BBa_C0062 BBa_c0062] and sequence alignment[1] (78.9% consensus and removed 3bp)
LuxR 753 1050941 1051693 789 1541 [http://biocyc.org/VFIS312309/NEW-IMAGE?type=GENE-IN-MAP&object=VFA0925 Biocyc]

[1]: Sequence Alignment with [http://biocyc.org/VFIS312309/sequence-delimited?chromosome=VF-CHROM-ES114-II&object=VFA0924&left=1050153&right=1051693 Biocyc Vibrio Fischeri (1050153-1051693)] (end of LuxI to end of LuxR).
[2]: Sequence Alignment with the reverse compliment of [1].

Related Biobricks

  • [http://partsregistry.org/Part:BBa_R0062 BBa_R0062] or [http://partsregistry.org/Part:BBa_R1062 BBa_R1062] in conjunction with or instead of new construct (pRE).
  • [http://partsregistry.org/Part:BBa_C0062 BBa_C0062] in conjunction with or instead of LuxR.
  • [http://partsregistry.org/Part:BBa_C0061 BBa_C0061] or [http://partsregistry.org/Part:BBa_C0161 BBa_C0161] in conjunction with or instead of LuxI.

Other interesting parts that may come in use (all working):

  • [http://partsregistry.org/Part:BBa_F2621 BBa_F2621]
  • [http://partsregistry.org/Part:BBa_F2622 BBa_F2622]
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]

Other interesting parts we may or may not use.

  • [http://partsregistry.org/Part:BBa_J37019 BBa_J37019] titled AHL induced LuxR generator.
  • [http://partsregistry.org/Part:BBa_J37032 BBa_J37032] if we want to test if [http://partsregistry.org/Part:BBa_R0062 BBa_R0062] works.
  • [http://partsregistry.org/Part:BBa_J37033 BBa_J37033] looks pretty useless.
  • [http://partsregistry.org/Part:BBa_I731012 BBa_I731012] looks interesting, but we're probably not going to do anything about it.

Problems

  1. Aligning [http://partsregistry.org/Part:BBa_R0062 BBa_R0062] and [http://partsregistry.org/Part:BBa_R1062 BBa_R1062] gives 92.2% consensus.
  2. Aligning [http://partsregistry.org/Part:BBa_R0063 pLUX-left] and [http://partsregistry.org/Part:BBa_R0062 pLUX-right] produced a 64% consensus.
    • Why are so many nucleotides different?
    • Why do the two promoters overlap so much (exactly according to the sequence alignment), yet the [http://partsregistry.org/Part:BBa_R0062:Design design] doesn't say so?
      • Are they using a difference Vibrio Fischeri strain for both?
  3. A sequence [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?tool=portal&db=protein&term=&query_key=1&dopt=gp&dispmax=20&page=1&qty=1&WebEnv=0NZu2wco5pgt4AFlKGBZSBuHz1xTMr4TRAfhLUXMT0Mg3JomrvvDqnSCvcYGDYXw1omCOSTlDgeb-UU%40264C254084F93770_0012SID&WebEnvRq=1 similar to LuxR] exists in the E. coli strain we're using. Don't know if it'll affect anything.

References

  • McMillen, D. et al (2002) "[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=117365 Synchronizing genetic relaxation oscillators by intercell signalling]", Proc Natl Acad Sci USA, Vol. 22, No. 3, pp. 679-684.
    • Same paper as the main paper from the oscillator section.


The Tuner

A way to control the oscillator's time period.

Components

Problems

  1. The CIIIC protein does not appear in any other papers (nor does it appear on pdb for that matter, but then again CIII doesn't either). So we have to believe that one paper.
  2. By synthesizing the CIIIC protein from DNA, we have to add the start codon ATG, resulting in an extra Methionine at the start. This may affect our experiments.

References

  • Hadler, S. et al (2007) [http://www.ncbi.nlm.nih.gov/pubmed/17890311 Probing the Anitprotease Activity of lCIII, an Inhibitor of the Escherichia coli Metalloprotease HflB (FtsH)]", Journal of Bacteriology, Vol. 189, No. 22, pp. 8130-8138.
    • The only paper about the experimental results of CIIIC. Also has a lot of experimental values (such as ratios) that can be used.
  • Herman, C. et al (1997) "[http://jb.asm.org/cgi/content/abstract/179/2/358 The HflB Protease of Escherichia coli Degrades Its Inhibitor λcIII]", Journal of Bacteriology, Vol. 179, No. 2, pp. 358-363.
    • Also has a lot of experimental values that could be used.

Sequences

This section lists the sequences of promoters and genes.

Promoters

The list of promoters used.

pRE

  • few hundred bp upstream from the CI gene.
  • 40 to 60bp long.
  • Appropriate Literature
    • [http://www.ncbi.nlm.nih.gov/pubmed/14872063?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Interactions among CII protein, RNA polymerase adn λ pRE promoter: contacts between RNA polymerase and the -35 region of pRE are identical in the presence and absence of CII protein]
    • [http://www.ncbi.nlm.nih.gov/pubmed/2953648?ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Mutations that improve the pRE promoter of coliphage lambda]
    • [http://www.ncbi.nlm.nih.gov/pubmed/2953649?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cross-specificities between cII-like proteins and pRE-like promoters of lambdoid bacteriophages] 
complement(38391->38340) on lambda phage.
AGAGCCTCGTTGCGTTTGTTTGCACGAACCATATGTAAGTATTTCCTT|AGAT (52bp)
                                                +----  transcription start site ---->
so what we want:
AGAGCCTCGTTGCGTTTGTTTGCACGAACCATATGTAAGTATTTCCTT (48bp)



pRE Overlaps with CII (but in the other direction)
                       ATGGTTCGTGCAAACAAACGCAACGAGGCTCTACGAATCGAGAGTGCGTTG--> CII cont'
<--ATCTAAGGAAATACTTACATATGGTTCGTGCAAACAAACGCAACGAGGCTCT (<--pRE)

For reference, [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=full_report&list_uids=3827059 CI]: 37940-37227



Primers:
AGAGCCTCGTTGCGTTT (len:17, tm:55deg, GC%:53)
AAGGAAATACTTACATATGGTTCGTG (len:26, tm:55deg, GC%:35)

Primer used regions:
AGAGCCTCGTTGCGTTTGTTTGCACGAACCATATGTAAGTATTTCCTT

There is only 5bp not included in the primer. Feels kinda weird.

pLUX

  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 Part:BBa_R0062-luxR & HSL regulated lux pR] 
acctgtaggatcgtacaggtttacgcaagaaaatggtttgttatagtcgaataaa(55 bp)

pLac

[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] (200bp)


Genes

The list of genes used.

CII

  • A λ phage gene.
  • [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NP_040630.1 NCBI protein]: 97aa
  • [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?WebEnv=0_IrKu54rUXySHgWXB0EaSRAhFNRuWQc8t_9Pvcu9deRGYI7MtHyfyf2B79C0DiodB6zisjQl5K8vOD%40264C254084F93770_0012SID&db=nucleotide&qty=1&c_start=1&list_uids=NC_001416.1&uids=&dopt=fasta&dispmax=5&sendto=&fmt_mask=0&from=38360&to=38653&extrafeatpresent=1&ef_CDD=8&ef_MGC=16&ef_HPRD=32&ef_STS=64&ef_tRNA=128&ef_microRNA=256&ef_Exon=512 NCBI nucleotide]: 294bp 

>gi|9626243:38360-38653 Enterobacteria phage lambda, complete genome
ATGGTTCGTGCAAACAAACGCAACGAGGCTCTACGAATCGAGAGTGCGTTGCTTAACAAAATCGCAATGC
TTGGAACTGAGAAGACAGCGGAAGCTGTGGGCGTTGATAAGTCGCAGATCAGCAGGTGGAAGAGGGACTG
GATTCCAAAGTTCTCAATGCTGCTTGCTGTTCTTGAATGGGGGGTCGTTGACGACGACATGGCTCGATTG
GCGCGACAAGTTGCTGCGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCC
AGATGGAGTTCTGA

Primers:
ATGGTTCGTGCAAACAAACG (56deg)
tcagaactccatctggatttgt

CIIIC

  • CIII (A λ phage gene) after residues 1-13 and 42-54 are cleaved (so only residues 14-41) are left.
  • Inhibites the degradation rate HtlB has on CII by binding to HtlB.
  • HtlB does not degrade CIIIC (unlike CIII).
  • [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&id=9626284 NCBI (CIII) protein]:54aa
  • [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?WebEnv=0cMBABYtiOTu-KvSSXngm5VVsgrz7RFiY24GdDY0xzrnBMiRgRDlrJjhu6Ybt2Q7bn2aiqpN4WI4qWl%40264C254084F93770_0012SID&db=nucleotide&qty=1&c_start=1&list_uids=NC_001416.1&uids=&dopt=fasta&dispmax=5&sendto=&from=33299&to=33463&strand=on NCBI (CIII) gene]:165bp

CIII gene: 

>gi|9626243:c33463-33299 Enterobacteria phage lambda, complete genome
ATGCAATATGCCATTGCAGGGTGGCCTGTTGCTGGCTGCCCTTCCGAATCTTTACTTGAACGAATCACCC
GTAAATTACGTGACGGATGGAAACGCCTTATCGACATACTTAATCAGCCAGGAGTCCCAAAGAATGGATC
AAACACTTATGGCTATCCAGACTAA

Start with 165bp (54 residues + stop codon) then cleave off residues 2-13 (39bp) and 42-54 (39bp), leaving residues 1,14-41,55 (90bp)

CIIIC (with added ATG at the start): 

ATGCAATATGCCATTGCAGGGTGGCCTGTTGCTGGCTGCCCTTCCGAATCTTTACTTGAACGAATCACCC
GTAAATTACGTGACGGATGGAAACGCCTTATCGACATACTTAATCAGCCAGGAGTCCCAAAGAATGGATC
AAACACTTATGGCTATCCAGACTAA

Changes to: 

ATGCCTTCCGAATCTTTACTTGAACGAATCACCC
GTAAATTACGTGACGGATGGAAACGCCTTATCGACATACTTAATCAGCCAGGA
TAA

The resulting primers are: 

ATGCCTTCCGAATCTTTACTTG
ttatcctggctgattaagtatgtcg (56deg)

HtlB

  • An E. coli gene.
  • HtlB degrades CII.
  • [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11506 Ecocyc]: 1935bp.
  • No extra EcoRI,PstI,SpeI,XbaI in gene below. 
>EG11506 hflB (complement(3324957..3323023)) E. coli
atgGCGAAAA ACCTAATACT CTGGCTGGTC ATTGCCGTTG TGCTGATGTC AGTATTCCAG
AGCTTTGGGC CCAGCGAGTC TAATGGCCGT AAGGTGGATT ACTCTACCTT CCTACAAGAG
GTCAATAACG ACCAGGTTCG TGAAGCGCGT ATCAACGGAC GTGAAATCAA CGTTACCAAG
AAAGATAGTA ACCGTTATAC CACTTACATT CCGGTTCAGG ATCCGAAATT ACTGGATAAC
CTGTTGACCA AGAACGTCAA GGTTGTCGGT GAACCGCCTG AAGAACCAAG CCTGCTGGCT
TCTATCTTCA TCTCCTGGTT CCCGATGCTG TTGCTGATTG GTGTCTGGAT CTTCTTCATG
CGTCAAATGC AGGGCGGCGG TGGCAAAGGT GCCATGTCGT TTGGTAAGAG CAAAGCGCGC
ATGCTGACGG AAGATCAGAT CAAAACGACC TTTGCTGACG TTGCGGGCTG CGACGAAGCA
AAAGAAGAAG TTGCTGAACT GGTTGAGTAT CTGCGCGAGC CGAGCCGCTT CCAGAAACTC
GGCGGTAAGA TCCCGAAAGG CGTCTTGATG GTCGGTCCTC CGGGTACCGG TAAAACGCTG
CTGGCGAAAG CGATTGCAGG CGAAGCGAAA GTTCCGTTCT TTACTATCTC CGGTTCTGAC
TTCGTAGAAA TGTTCGTCGG TGTGGGTGCA TCCCGTGTTC GTGACATGTT CGAACAGGCG
AAGAAAGCGG CACCGTGCAT CATCTTTATC GATGAAATCG ACGCCGTAGG CCGCCAGCGT
GGCGCTGGTC TGGGCGGTGG TCACGATGAA CGTGAACAGA CTCTGAACCA GATGCTGGTT
GAGATGGATG GCTTCGAAGG TAACGAAGGT ATCATCGTTA TCGCCGCGAC TAACCGTCCG
GACGTTCTCG ACCCGGCCCT GCTGCGTCCT GGCCGTTTCG ACCGTCAGGT TGTGGTCGGC
TTGCCAGATG TTCGCGGTCG TGAGCAGATC CTGAAAGTTC ACATGCGTCG CGTACCATTG
GCACCCGATA TCGACGCGGC AATCATTGCC CGTGGTACTC CTGGTTTCTC CGGTGCTGAC
CTGGCGAACC TGGTGAACGA AGCGGCACTG TTCGCTGCTC GTGGCAACAA ACGCGTTGTG
TCGATGGTTG AGTTCGAGAA AGCGAAAGAC AAAATCATGA TGGGTGCGGA ACGTCGCTCC
ATGGTGATGA CGGAAGCGCA GAAAGAATCG ACGGCTTACC ACGAAGCGGG TCATGCGATT
ATCGGTCGCC TGGTGCCGGA ACACGATCCG GTGCACAAAG TGACGATTAT CCCACGCGGT
CGTGCGCTGG GTGTGACTTT CTTCTTGCCT GAGGGCGACG CAATCAGCGC CAGCCGTCAG
AAACTGGAAA GCCAGATTTC TACGCTGTAC GGTGGTCGTC TGGCAGAAGA GATCATCTAC
GGGCCGGAAC ATGTATCTAC CGGTGCGTCC AACGATATTA AAGTTGCGAC CAACCTGGCA
CGTAACATGG TGACTCAGTG GGGCTTCTCT GAGAAATTGG GTCCACTGCT GTACGCGGAA
GAAGAAGGTG AAGTGTTCCT CGGCCGTAGC GTAGCGAAAG CGAAACATAT GTCCGATGAA
ACTGCACGTA TCATCGACCA GGAAGTGAAA GCACTGATTG AGCGTAACTA TAATCGTGCG
CGTCAGCTTC TGACCGACAA TATGGATATT CTGCATGCGA TGAAAGATGC TCTCATGAAA
TATGAGACTA TCGACGCACC GCAGATTGAT GACCTGATGG CACGTCGCGA TGTACGTCCG
CCAGCGGGCT GGGAAGAACC AGGCGCTTCT AACAATTCTG GCGACAATGG TAGTCCAAAG
GCTCCTCGTC CGGTTGATGA ACCGCGTACG CCGAACCCGG GTAACACCAT GTCAGAGCAG
TTAGGCGACA AGTAA

HtlB primers:
atgGCGAAAAACCTAATACTCTG
ttacttgtcgcctaactgct

LuxI

  • A Vibrio Fischeri gene.
  • [http://biocyc.org/VFIS312309/NEW-IMAGE?type=GENE&object=VFA0924 Biocyc]: 573bp
  • Biobrick available
    • [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37034 BBa_J37034]: LuxI + GFP. We could use this if we can extract the RBS+LuxI part. 
>VFA0924 VFA0924 (complement(1050725..1050153)) V. fischeri chromosome es114 ii
ATGataaaaa aatcggactt tttgggcatt ccatcagagg agtatagagg tattcttagt
cttcgttatc aggtatttaa acgaagactg gagtgggact tggtaagtga ggataatctt
gaatcagatg aatatgataa ctcaaatgca gaatatattt atgcttgtga tgatgcggaa
gaggtaaatg gctgttggcg tttgttacct acaacgggtg attacatgtt aaaaactgtt
tttcctgaat tgctcggaga tcaagtagcc ccaagagatc caaatatagt cgaattaagc
cgttttgctg tgggaaaaaa tagctcaaaa ataaataact ctgctagtga aataacaatg
aaattgtttc aagctatata taaacacgca gttagtcaag gtattacaga atatgtaaca
gtaacatcaa tagcaataga gcgatttctg aaacgtatta aagttccttg tcatcgcatt
ggtgataagg agattcattt attaggtaat actagatctg ttgtattgtc tatgcctatt
aatgatcagt ttagaaaagc tgtatcaaat taa

LuxI primers:
ATGataaaaaaatcggactttttggg
ttaatttgatacagcttttctaaactgatc (length=30 too long?)

LuxR

  • A Vibrio Fischeri gene.
  • [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3280300&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum NCBI]: 753bp.
  • Biobrick available
    • [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37019 BBa_J37019]: Promoter + LuxR. 862bp 
>VFA0925 VFA0925 1050941..1051693 V. fischeri chromosome es114 ii
ATGaacatta aaaatataaa tgctaatgag aagataattg ataaaattaa aacttgtaat
aataataaag atattaatca atgtttatct gaaatagcaa agataataca ttgtgaatat
tacctattcg ctattatcta tcctcactca ataattaaac ctgatgtttc aattatagat
aattaccctg aaaaatggcg taaatattat gatgatgccg gactactaga atatgaccct
gtagtcgatt actctaagtc ccatcattca ccaattaatt ggaacgtatt cgaaaaaaaa
acaataaaaa aagagtctcc gaatgtaata aaagaagcac aggaatcggg actcattact
ggatttagct ttccaattca tactgcaagt aatggttttg gaatgctcag ttttgctcat
tcagataaag atatttatac tgacagttta tttttacacg ctagtacaaa tgtaccatta
atgcttcctt ctttagtcga taattatcaa aaaataaata cgacacgtaa aaagtcagat
tctattttaa caaaaagaga aaaagaatgc ttagcgtggg cgagtgaagg aaaaagtaca
tgggatattt caaaaatact tggctgcagt gagcgtactg tcacttttca tttaaccaat
actcaaatga aactcaatac aactaaccgc tgccaaagta tttctaaagc aattttaact
ggcgccatta attgtccata ccttaaaaat taa

LuxR primers:
ATGaacattaaaaatataaatgctaatgagaaga (length=34 too long?)
ttaatttttaaggtatggacaattaatggc (length=30 too long?)


Testing

How we plan to test our parts when we've finished constructed them.

Promoter Testing

NYMU reloxilator promoter testing.png

We are attempting to establish a "inducible promoter standard," much like the repressible promoter standard, where instead we define the IPTG inducible "standard" pLac promoter as having a value of 1, while all other inducible promoters will be measured in multiples of this standard. Then the value of IPTG can also be varied to test the ratios between the promoters as to whether the ratio is linear, sinusoidal, polynomial, etc.

We can test pRE promoter via this standard by connecting the |CII gene behind pLac, and connecting a reporter behind pRE.

This standard can also be used to test promoters that require a complex to be induced, such as pLux. pLux requires the R+AHL complex to be induced. To test the the strength of pLux, first let all parts of the complex save one to be always existing, while the last one would be controlled by pLac. In this example, LuxR is always expressed and the amount of AHL is determined by pLac.

Component Testing

NYMU reloxilator component testing.png

This is the part where we test to see if our component works. The diagram above shows the pathway between the various inputs, and the output (GFP).

Modelling

Oscillator Modelling 1

NYMU reloxilator modelling.png

  • HtlB concentration
  • CII concentration.
  • 20sec intervals.

Variables Used

  • pRE Stength: 0.4 (1 CII protein produces 0.4 CII and HtlB proteins).
  • H-t1/2: 8mins (random value I picked, its probably not the real value.)
    • Half life of HtlB.
    • Changing this value just makes the oscllation period longer.
  • C0: 8mins.
    • CII t1/2 with no HtlB present. Based on [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=94496 Shotland2000])
  • C1: 1min.
    • Also based on the same paper.
    • CII t1/2 with an equal amount of HtlB is what I assume because they don't say.
  • Δt: 20sec
    • Time segment used for the calculation (can't be too close to the t1/2's).

Formulas used

The HtlB/CII ratio used to calculate the t1/2 at a specific time point: 

R = [HtlB] / [CII] * [CII] = [HtlB]     ----- (1)

The last *[CII] has something to do with the [HtlB][CII] --> [CII] equation.

Calculation of CII's t1/2 based on the ratio above: 

C-t1/2 = C0 x (C1/C0)R     ----- (2)

The calculation of amount of substrates remaining w.r.t the half lives: 

[HtlB] = [HtlB] x 0.5(Δt / H-t1/2)     ----- (3)

[CII] = [CII] x 0.5(Δt / C-t1/2)     ----- (4)

Conclusion

Fiddling around a bit yields:

Variable Affects
Initial concentration of: HtlB (higher) (longer) startup time
CII (higher)
Natural degradation rate of HtlB (longer) (longer) time period
CII (longer)
pRE strength (higher) (shorter) time period
HtlB to CII degradation rate (higher) (lower) global maximum concentration (not counting startup)

Is a promoter that can be activated by HtlB? because then our calculations would be even more accurate.

Experimental Results

Reloxilator construct schedule.png

This is a picture of all the constructs, and green constructs are the ones that were successfully created.

References

  1. McMillen, D. et al (2002) "[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=117365 Synchronizing genetic relaxation oscillators by intercell signalling]", Proc Natl Acad Sci USA, Vol. 22, No. 3, pp. 679-684.
  2. Hadler, S. et al (2007) [http://www.ncbi.nlm.nih.gov/pubmed/17890311 Probing the Anitprotease Activity of λCIII, an Inhibitor of the Escherichia coli Metalloprotease HflB (FtsH)]", Journal of Bacteriology, Vol. 189, No. 22, pp. 8130-8138.

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