Team:HKUST-Hong Kong/notebook.html

From 2011.igem.org

(Difference between revisions)
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<ul>
<ul>
   <li>λ RED: RFP with homologous sequence PCR successful</li>
   <li>λ RED: RFP with homologous sequence PCR successful</li>
-
   <li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished</li>
+
   <li>2010 Slovenia’s method - CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished</li>
   <li>Split superfolder GFP system : PCR of spilt superfolder GFP successful</li>
   <li>Split superfolder GFP system : PCR of spilt superfolder GFP successful</li>
   <li>2010 Slovenia’s method - CFP/YFP :Ligation with promoter is  successful, but cannot see the green fluorescence, considering redo</li>
   <li>2010 Slovenia’s method - CFP/YFP :Ligation with promoter is  successful, but cannot see the green fluorescence, considering redo</li>
Line 161: Line 161:
   <li>pir  gene: Sequencing product did not meet sequencing requirement – sequencing  rejected</li>
   <li>pir  gene: Sequencing product did not meet sequencing requirement – sequencing  rejected</li>
   <li>Split superfolder GFP system: Ligation product transformed was not  as expected. Low recovery from gel purification</li>
   <li>Split superfolder GFP system: Ligation product transformed was not  as expected. Low recovery from gel purification</li>
-
   <li>2010 Slovenia’s method - CFP/YFP: combination of n-terminal and c-terminal CDS onto same plasmid, driven  by lac promoter of pBluescriptKS+ completed à no fluorescence</li>
+
   <li>2010 Slovenia’s method - CFP/YFP: combination of n-terminal and c-terminal CDS onto same plasmid, driven  by lac promoter of pBluescriptKS+ completed, very weak fluorescence</li>
   <li><em>nadE</em> gene: successful completion of operon with terminator with biobrick digestion,  component is putatively finished as biobrick</li>
   <li><em>nadE</em> gene: successful completion of operon with terminator with biobrick digestion,  component is putatively finished as biobrick</li>
   <li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be  successful. Repeating fusion PCR</li>
   <li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be  successful. Repeating fusion PCR</li>
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   <ul>
   <ul>
     <li>2~4 folded increasing for Kan</li>
     <li>2~4 folded increasing for Kan</li>
-
     <li>Proton gradient (H+) driven </li>
+
     <li>Proton gradient (H<sup>+</sup>) driven </li>
     <li>Pumps out other toxins</li>
     <li>Pumps out other toxins</li>
     <li>Unknown promoter </li>
     <li>Unknown promoter </li>
   </ul>
   </ul>
-
   <li><em>E. coli</em> DH10a containing pUC18not/T4MO arrived. </li>
+
   <li><em>E. coli</em> DH10a containing pUC18Not/T4MO arrived. </li>
</ul>
</ul>
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th  Aug</strong><strong>)</strong><strong> </strong><br>
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th  Aug</strong><strong>)</strong><strong> </strong><br>
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   <li>pir  gene: Sequencing result has just come out</li>
   <li>pir  gene: Sequencing result has just come out</li>
   <li>Split superfolder GFP system: Finished ligation of lacI promotor and  GFP 11 and verifying. GFP 1-10 PCRing</li>
   <li>Split superfolder GFP system: Finished ligation of lacI promotor and  GFP 11 and verifying. GFP 1-10 PCRing</li>
-
   <li>2010 Slovenia’s method- CFP/YFP: Finished construction but not verified</li>
+
   <li>2010 Slovenia’s method - CFP/YFP: Finished construction but not verified</li>
   <li><i>nadE</i>  gene: finished</li>
   <li><i>nadE</i>  gene: finished</li>
   <li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li>
   <li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li>
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   <li>Completed the standard curve for  OD 600 versus RFP/KanR CFU concentration</li>
   <li>Completed the standard curve for  OD 600 versus RFP/KanR CFU concentration</li>
   <li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li>
   <li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li>
-
   <li>Successfully extracted T4MO  from pUC18not/T4MO, discovering the inclusion of a native constitutive  promoter, and ligated to pBlueScript KS+ to create a SpeI site for biobrick  assembly. </li>
+
   <li>Successfully extracted T4MO  from pUC18Not/T4MO, discovering the inclusion of a native constitutive  promoter, and ligated to pBlueScript KS+ to create a SpeI site for biobrick  assembly. </li>
   <li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li>
   <li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li>
</ul>
</ul>
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   <li>pir  gene: Exact location of pir gene in BW25141 is mapped out</li>
   <li>pir  gene: Exact location of pir gene in BW25141 is mapped out</li>
   <li>Split  superfolder GFP system: Primers have problem.  Waiting for new primers to come next week</li>
   <li>Split  superfolder GFP system: Primers have problem.  Waiting for new primers to come next week</li>
-
   <li>2010 Slovenia’s method- CFP/YFP: CFP ligated with pET. YFP constructing</li>
+
   <li>2010 Slovenia’s method - CFP/YFP: CFP ligated with pET. YFP constructing</li>
   <li>oriR101&amp;repA101-ts:  Verifying oriR101&amp;repA101-ts </li>
   <li>oriR101&amp;repA101-ts:  Verifying oriR101&amp;repA101-ts </li>
   <li>λ RED: PCR with the new primers is successful. Modified protocol using  KAN-resistance gene to swap out uidA gene</li>
   <li>λ RED: PCR with the new primers is successful. Modified protocol using  KAN-resistance gene to swap out uidA gene</li>
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<ul>
<ul>
   <li>Digestion of T4MO/pBS KS+ failed</li>
   <li>Digestion of T4MO/pBS KS+ failed</li>
-
   <li>Successfully ligated bcr gene with RBS (later confirmed to be false positive) </li>
+
   <li>Successfully ligated <i>bcr</i> gene with RBS (later confirmed to be false positive) </li>
</ul>
</ul>
<p><strong>Week 10 (15th-19th  Aug) </strong><br>
<p><strong>Week 10 (15th-19th  Aug) </strong><br>
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   <li>pir gene: ligation of pir gene  and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of  pBluescriptKS+ backbone<strong></strong></li>
   <li>pir gene: ligation of pir gene  and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of  pBluescriptKS+ backbone<strong></strong></li>
   <li>Split  superfolder GFP  system: Re-digestion  and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during  transformation<strong> </strong></li>
   <li>Split  superfolder GFP  system: Re-digestion  and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during  transformation<strong> </strong></li>
-
   <li>2010 Slovenia’s method- CFP/YFP: digestion  and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.<strong> </strong></li>
+
   <li>2010 Slovenia’s method - CFP/YFP: digestion  and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.<strong> </strong></li>
   <li><em>nadE</em> gene: Complete.<strong> </strong></li>
   <li><em>nadE</em> gene: Complete.<strong> </strong></li>
   <li>oriR101&amp;repA101-ts: ligation of oriR101, repA101 and the backbone  pSA1K3 in process; transformation  result available tomorrow; colony PCR of λ red done, failed.<strong> </strong></li>
   <li>oriR101&amp;repA101-ts: ligation of oriR101, repA101 and the backbone  pSA1K3 in process; transformation  result available tomorrow; colony PCR of λ red done, failed.<strong> </strong></li>
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<ul>
<ul>
   <li>pir gene: Background self-ligation is under test,  results will be available tomorrow </li>
   <li>pir gene: Background self-ligation is under test,  results will be available tomorrow </li>
-
   <li>Split  superfolder GFP system: Background self-ligation is under test,  results will be available tomorrow; Ligating split GFP  with <em>lac</em>promoter </li>
+
   <li>Split  superfolder GFP system: Background self-ligation is under test,  results will be available tomorrow; Ligating split GFP  with <em>lac</em> promoter </li>
-
   <li>2010 Slovenia’s method- CFP/YFP: pET_CFP and pET_YFP  have been constructed and verified; transformation of  each into BL21 has been done; </li>
+
   <li>2010 Slovenia’s method - CFP/YFP: pET_CFP and pET_YFP  have been constructed and verified; transformation of  each into BL21 has been done; </li>
-
   <li><em>nadE</em> gene: Completed; veri; </li>
+
   <li><em>nadE</em> gene: Completed; verified; </li>
-
   <li>oriR101 + repA101ts: Construction is complete – verified by  restriction digestion; Biobrick currently  located on pSB1AK3; </li>
+
   <li>oriR101 + repA101ts: Construction is complete – verified by  restriction digestion; BioBrick currently  located on pSB1AK3; </li>
   <li>λ RED:check whether swap is successful: screen 6  colonies for verification; </li>
   <li>λ RED:check whether swap is successful: screen 6  colonies for verification; </li>
   <li>pToolkit construction: Complete </li>
   <li>pToolkit construction: Complete </li>
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   <li>Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)</li>
   <li>Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)</li>
   <li>Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed] </li>
   <li>Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed] </li>
-
   <li>Started to construct Biobrick of  bcr gene for submission</li>
+
   <li>Started to construct BioBrick of  bcr gene for submission</li>
</ul>
</ul>
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br>
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br>
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<ul>
<ul>
   <li>oriR101&amp;repA101-ts: the progress is not  ideal, cannot finishthe characterizationthis week </li>
   <li>oriR101&amp;repA101-ts: the progress is not  ideal, cannot finishthe characterizationthis week </li>
-
   <li>pir gene: sequence result: some parts is missing (the target part, for an unexpected cut on that –illegal cut (point mutation)  or star activity; insert the pir to pBS again (use different enzymes), sequence again. </li>
+
   <li>pir gene: sequence result: some key parts are missing. the target part developed an unexpected illegal cut (point mutation or star activity); insert the pir to pBS again (using different enzymes), sequenced again. </li>
   <li>Split superfolder GFP system: the construction of split GFP+backbone finished; characterization in  progress </li>
   <li>Split superfolder GFP system: the construction of split GFP+backbone finished; characterization in  progress </li>
</ul>
</ul>

Revision as of 18:57, 5 October 2011


Notebook




Week 1 (4th-10th June)
Strain construction:

  • Genomic DNA of E. coli DH10b extracted
  • Waiting for materials to arrive

Week 2 (13rd-17th June)
Strain construction:

  • Genomic DNA of E. coli DH10b extracted Genomic DNA of BL21 extracted
  • Secured split superfolder GFP construct transformed out
  • Finished design of PCR primers

Culture Tests:

  • Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)
  • Constructed standard curve for OD600 verses RR1 CFU concentration

Week 3 (20th-24th June)
Strain construction:

  • nadE: PCR out nadE gene from the genome of BL21
  • Split superfolder GFP system: The test of the intact superfolder GFP is successful. Experiments will be conducted soon.
  • 2010 Slovenia’s method - CFP/YFP: BioBricks are transformed into E. coli DH10b. Test will be conducted soon.
  • λ RED and oriR101&repA101-ts: pKD46 has arrived and successful extracted. RFP reporter system is ready. Primers of RepA101ts-OriR101 are ready.

Culture Tests:

  • Performed 2nd and 3rd MIC test for wild type (kanamycin gradient 5-20 µg/ml, 2µg/ml intervals)
  • Miniprep of BBa_I763007 and BBa_E1010 was successful

Week 4 (27th June–1st July)
Strain construction:

  • λ RED : protocol design finished, pKD46 arrived and digestion test was successful, PCR RFP with homologous sequence was successful
  • 2010 Slovenia’s method - CFP/YFP: protocol design finished, successfully finished combined CFP and YFP
  • Split superfolder GFP system: protocol design finished, primer arrived
  • Pir gene and ori-γ: protocol under construction, primer for ori-γ arrived, BW25141 gDNA extraction successful

Culture Tests:

  • Ligation of pSB2K3 (from BBa_E1010) with RFP report device (BBa_I763007)
  • Transformation of the RFP/KanR plasmid to E. coli DH10b

Week 5 (8th-12th July)
Strain construction:

  • ori-γ: Primers arrived, PCR was successful. Result: one of four samples survived, but of low concentration
  • pir gene: gDNA of BW25141 extracted
  • Split superfolder GFP system: GFP1-10 PCRed, GFP11 PCRed
  • 2010 Slovenia’s method - CFP/YFP: Verified combined fluorescence protein
  • oriR101&repA101-ts: PCR was successful

Culture Tests:

  • Successful construction of a RFP-labeled kanamycin-resistant strain .
  • Literature search on mechanisms to raise the MIC for the proposed T4MO mutant slightly to help it survive in kanamycin long enough to fulfill its function.
  • MIC testing for RR-1

Week 6 (15th-19th July)
Strain construction:

  • λ RED: RFP with homologous sequence PCR successful
  • 2010 Slovenia’s method - CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished
  • Split superfolder GFP system : PCR of spilt superfolder GFP successful
  • 2010 Slovenia’s method - CFP/YFP :Ligation with promoter is successful, but cannot see the green fluorescence, considering redo
  • pToolkit construction: PCR of ori-γ successful, ligation with pKD46 backbone successful, wait to do colony PCR to check the existence of ori-γ, the sequence PCR of the pir gene is done, wait to check the result
  • nadE gene: ligate nadE gene with double terminator, not successful, will repeat experiment

Culture Tests:

  • MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals)
  • MIC test for mixed cultures of RFP/KanR and RR1(1:99)
  • Literature search – multidrug pump candidates
    • Bcr(~1.2kbp): overexpression increases kanamycin MIC ~2-4fold
    • NorM (~1.3kbp): over expression reduces radical oxidative species (e.g. H2O2) inside the cell

Week 7 (22nd-26th July)
Strain construction:

  • pir gene: Sequencing product did not meet sequencing requirement – sequencing rejected
  • Split superfolder GFP system: Ligation product transformed was not as expected. Low recovery from gel purification
  • 2010 Slovenia’s method - CFP/YFP: combination of n-terminal and c-terminal CDS onto same plasmid, driven by lac promoter of pBluescriptKS+ completed, very weak fluorescence
  • nadE gene: successful completion of operon with terminator with biobrick digestion, component is putatively finished as biobrick
  • oriR101&repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR
  • λ RED: Previous experiment of gene swapping failed. Trouble-shooting in progress
  • pToolkit construction: results from colony PCR of ori-γ from transformed bacteria: successful completion of pToolkit

Culture Tests:

  • Mixed culture MIC tests for RFP/KanR and RR1(1:99)
  • Multidrug Efflux Pump – Settled on Bcr as the candidate gene
    • 2~4 folded increasing for Kan
    • Proton gradient (H+) driven
    • Pumps out other toxins
    • Unknown promoter
  • E. coli DH10a containing pUC18Not/T4MO arrived.

Week 8 (1st-5th Aug)
Strain construction:

  • pir gene: Sequencing result has just come out
  • Split superfolder GFP system: Finished ligation of lacI promotor and GFP 11 and verifying. GFP 1-10 PCRing
  • 2010 Slovenia’s method - CFP/YFP: Finished construction but not verified
  • nadE gene: finished
  • oriR101&repA101-ts: Been ligated to a backbone, verifying
  • λ RED: Waiting for the primers to construct the linear sequence

Culture Tests:

  • Completed the standard curve for OD 600 versus RFP/KanR CFU concentration
  • Mixed culture MIC tests for RFP/KanR and RR1(1:99)
  • Successfully extracted T4MO from pUC18Not/T4MO, discovering the inclusion of a native constitutive promoter, and ligated to pBlueScript KS+ to create a SpeI site for biobrick assembly.
  • PCR amplified bcr gene from gDNA of E. coli stock

Week 9 (8th-12th Aug)
Strain construction:

  • pir gene: Exact location of pir gene in BW25141 is mapped out
  • Split superfolder GFP system: Primers have problem. Waiting for new primers to come next week
  • 2010 Slovenia’s method - CFP/YFP: CFP ligated with pET. YFP constructing
  • oriR101&repA101-ts: Verifying oriR101&repA101-ts
  • λ RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene
  • pCarrier: MCS is hybridized. pSB1K3 is under digestion

Culture Tests:

  • Digestion of T4MO/pBS KS+ failed
  • Successfully ligated bcr gene with RBS (later confirmed to be false positive)

Week 10 (15th-19th Aug)
Strain construction

  • pir gene: Ligation done and being verified
  • Split superfolder GFP system: PCR with new primers. Split superfolder GFP11 digested and ligated with the promoter.
  • λ RED: The swapping seemed to be successful
  • oriR101&repA101-ts: Waiting for new primers
  • pCarrier: MCS and OriR ligated

Culture Tests:

  • Indole MIC test for wild type (1mM with kanamycin gradient):
  • Successfully ligated T4MO into pBS KS+

Week 11 (22nd-26th Aug)
Strain construction:

  • pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone
  • Split superfolder GFP system: Re-digestion and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during transformation
  • 2010 Slovenia’s method - CFP/YFP: digestion and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.
  • nadE gene: Complete.
  • oriR101&repA101-ts: ligation of oriR101, repA101 and the backbone pSA1K3 in process; transformation result available tomorrow; colony PCR of λ red done, failed.
  • pToolkit construction: Complete
  • pCarrier: Ligation of MCS to nadE in pSB1AK3 is complete; Digestion check showed negative result; Hybridization of MCS in progress

Culture Tests:

  • Indole MIC test (500µM with kanamycin gradient)
  • Ligation of the RBS+Bcr with pLac promoter failed

Week 12 (29th Aug-1st Sep)
Strain Construction

  • pir gene: Background self-ligation is under test, results will be available tomorrow
  • Split superfolder GFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with lac promoter
  • 2010 Slovenia’s method - CFP/YFP: pET_CFP and pET_YFP have been constructed and verified; transformation of each into BL21 has been done;
  • nadE gene: Completed; verified;
  • oriR101 + repA101ts: Construction is complete – verified by restriction digestion; BioBrick currently located on pSB1AK3;
  • λ RED:check whether swap is successful: screen 6 colonies for verification;
  • pToolkit construction: Complete
  • pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS

Culture Tests

  • Mixed culture MIC tests for RFP/KanR and RR1 (1:99)
  • Indole MIC test (300µM with kanamycin gradient)
  • Successfully ligated T4MO with GFP

Week 13 (5th-9th Sep)
Strain construction

  • λ RED : previous PCR verification (S2) not show very clear result, halted for this week; new verificationprimers (S2) arrived
  • oriR101&repA101-ts: Construction of oriR101+pSB1AK2 successful; oriR101+pSB1Cs (standard BioBrick format) ligation done, colony PCR checked, digestion test tmr
  • Spilt superfolder GFP system: 2010 Slovenia’s method; split superfolder GFP from Biobrick.
  • pir gene and ori-γ: ligationof pir gene and pBluescriptKS+done, but do not have clear verification result (colony PCR+, digestion test-); considering new verification test (2 new sets).
  • nadE gene: Completed; wait to do sequencing verification;
  • pCarrier: MCS reinsert, change the size and position of insertion;
  • pToolkit construction: accidentally disappear, redo the whole plasmid;
  • pCarrier: nadE part ready, working on MCS now.

Culutre Test

  • Mixed culture MIC tests for RFP/KanR and RR1 (1:99)
  • Indole MIC test (1mM indole concentration with kanamycin gradient)
  • Successfully ligated T4MO/GFP into kanamycin resistant backbone.

Week 14 (13rd-17th Sep)
Strain Construction:

  • λ RED:basically successful; new S2 primers arrived, first trial failed (negative control of pKD46+E. coli DH10B still have some bands); consider directly PCR out from pKD46
  • oriR101&repA101-ts: constructionof oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of heat sensitivity in progress
  • Spilt superfolder GFP system: 2010 Slovenia’s method; split superfolder GFP from Biobrick;
  • pir gene and ori-γ: Pir ligation with pBS successful, ready for sequencing;
  • nadE gene: Completed; wait to do sequencing verification
  • pToolkit construction: accidentally lost, redo the whole thing
  • pCarrier : MCS insertion does not show good result halted for this year

Culture Test

  • Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)
  • Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed]
  • Started to construct BioBrick of bcr gene for submission

Week 15 (20th-24th Sep)
Strain Construction;

  • oriR101&repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week
  • pir gene: sequence result: some key parts are missing. the target part developed an unexpected illegal cut (point mutation or star activity); insert the pir to pBS again (using different enzymes), sequenced again.
  • Split superfolder GFP system: the construction of split GFP+backbone finished; characterization in progress

Week 16 ( 27th-30th Sep)
Strain Construction

  • oriR101-ts: have already submitted and received by part registry; rough characterization successful; further characterization method confirmed;
  • Split superfolder GFP system: ligation GFP1-10 and GFP11 into one plasmid finished, but not have fluorescence; starting to insert GFP1-10 in pSB1C3, GFP11 in pSB1AK3; then use two antibiotics as selection markers, then check the fluorescence
  • pir gene: sequence failed (wrong gene…nadE actually)
  • pToolkit construction: construction in progress




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Master List & Characterization Data

Human Practice

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