Team:British Columbia/Notebook/Labbook

From 2014.igem.org

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<!-- =================START Accordion Menu ======================-->
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<!-- ==================== ORTHOGONAL START ==================== -->
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               <a href="#oct_w2">Week 2</a><br>
               <a href="#oct_w2">Week 2</a><br>
               <a href="#oct_w3">Week 3</a><br>
               <a href="#oct_w3">Week 3</a><br>
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              <a href="#oct_w4">Week 4</a><br>
 
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<!-- ===================== END OF ORTHOGONAL ==================== -->
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<!-- ================ THIS IS BIOMINING ============================ -->
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        Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.
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            <h4>August</h4>
 +
              <a href="#b_aug_w3">Week 3</a><br>
 +
              <a href="#b_aug_w4">Week 4</a><br>   
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<h4>September</h4> 
 +
            <a href="#b_sept_w1">Week 1</a><br>
 +
              <a href="#b_sept_w2">Week 2</a><br>   
 +
            <a href="#b_sept_w3">Week 3</a><br>
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              <a href="#b_sept_w4">Week 4</a><br>   
       </div>
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<!-- ================== END OF BIOMINING ========================== -->
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<!-- =================END of Accordion Menu ======================-->
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<!-- =============== LAB NOTE BOOK STARTS ======================== -->
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<!-- =============== ORTHOGONAL LAB NOTE BOOK STARTS ======================== -->
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           <div class="col-md-8 pre-scrollable" script="max-height: 1000px">
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           <div class="col-md-8">
                 <h1 id="june_w3"> June - Week 3</h1>                 
                 <h1 id="june_w3"> June - Week 3</h1>                 
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<b> Experimenter </b>: Dan Korvin, Wenchen Zhao, Zeki Ekmekci
<b> Experimenter </b>: Dan Korvin, Wenchen Zhao, Zeki Ekmekci
  </p>  <p>
  </p>  <p>
-
<b> Aim </b> : Amplify T7 RNA polymerase gene (RNAP) and terminator gene by PCR.
+
<b> Aim </b> : Amplify T7 RNA polymerase gene (RNAP) from T7 bacteriophage genome and terminator from part BBa_B0015 by PCR.
  </p>  <p>
  </p>  <p>
<b> Result </b>: PCR was successful. Target bands were seen on the agarose gel.
<b> Result </b>: PCR was successful. Target bands were seen on the agarose gel.
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<b> Experimenter </b>: Zeki Ekmekci, Wenchen Zhao, Dan Korvin
<b> Experimenter </b>: Zeki Ekmekci, Wenchen Zhao, Dan Korvin
</p> <p>
</p> <p>
-
<b> Aim </b>: Amplify T7 DNA polymerase (DNAP), primase/helicase (P/H), single strand binding protein (SSBP) genes by PCR. Digest terminator with EcoRI (E) and XbaI (X). Digest RNAP with E and SpeI (S).
+
<b> Aim </b>: Amplify T7 DNA polymerase (DNAP), primase/helicase (P/H), single strand binding protein (SSBP) genes from T7 bacteriophage genome by PCR. Digest terminator with EcoRI (E) and XbaI (X). Digest RNAP with E and SpeI (S).
</p> <p>
</p> <p>
<b> Results </b>: NA.
<b> Results </b>: NA.
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<b> Experimenter </b>: Wenchen Zhao, Dan Korvin, Zeki Ekmekci
<b> Experimenter </b>: Wenchen Zhao, Dan Korvin, Zeki Ekmekci
</p><p>
</p><p>
-
<b> Aim </b>: Verify DNAP and P/H constructs by colony PCR. Digest SSBP and RNAP constructs with E and S. Digest Promoter + RBS plasmid with E and X. Transform the ligation products to E.coli.  
+
<b> Aim </b>: Verify DNAP and P/H constructs by colony PCR. Digest SSBP and RNAP constructs with E and S. Digest the plasmid containing Pbad promoter and RBS with E and X. Transform the ligation products to E.coli.  
</p><p>
</p><p>
<b> Results </b>: Colony PCR failed.  
<b> Results </b>: Colony PCR failed.  
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<b> Aim </b>: Verify P/H and DNAP constructs using colony PCR. Analyze sequencing results of SSBP and RNAP complete constructs.
<b> Aim </b>: Verify P/H and DNAP constructs using colony PCR. Analyze sequencing results of SSBP and RNAP complete constructs.
</p><p>
</p><p>
-
<b> Results </b>: Sequencing results are negative – no promoter/RBS found in SSBP and RNAP constructs. Colony PCR result was also negative.
+
<b> Results </b>: Sequencing results are negative – no Pbad promoter/RBS found in SSBP and RNAP constructs. Colony PCR result was also negative.
</p>
</p>
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<b> Experimenter </b>: Dan Korvin, Wenchen Zhao
<b> Experimenter </b>: Dan Korvin, Wenchen Zhao
</p><p>
</p><p>
-
<b> Aim </b>: Double digest DNAP, RNAP and SSBP constructs with X and PstI (P). Ligate three digested genes into RBS+Promoter plasmid digested with S and P. Miniprep P/H construct.
+
<b> Aim </b>: Double digest DNAP, RNAP and SSBP constructs with X and PstI (P). Ligate three digested genes into RBS + Pbad Promoter plasmid digested with S and P. Miniprep P/H construct.
</p><p>
</p><p>
<b> Results </b>: NA.
<b> Results </b>: NA.
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<b> Aim </b>: Analyze sequencing results for P/H construct. Digest P/H with E and S and digest 3-gene assembly with E and X. Ligate double digest P/H into double digested 3-gene vector. Transform the ligation product to E.coli.
<b> Aim </b>: Analyze sequencing results for P/H construct. Digest P/H with E and S and digest 3-gene assembly with E and X. Ligate double digest P/H into double digested 3-gene vector. Transform the ligation product to E.coli.
</p><p>
</p><p>
-
<b> Results </b>: Sequencing result was negative. Promoter/RBS was not present in the sequence. But XbaI restriction site was successfully mutated.
+
<b> Results </b>: Sequencing result was negative. Pbad romoter/RBS was not present in the sequence. But XbaI restriction site was successfully mutated.
</p>
</p>
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</p>
</p>
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<h1 id="oct_w1"> October - Week 2 </h1>
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<h1 id="oct_w2"> October - Week 2 </h1>
<h3> October 4th </h3>
<h3> October 4th </h3>
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</p>
</p>
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<h1 id="oct_w1"> October - Week 3 </h1>
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<h1 id="oct_w3"> October - Week 3 </h1>
<h3> October 14th </h3>
<h3> October 14th </h3>
<p>
<p>
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<b> Results </b>: DNAP and SSBP protein was successfully expressed. P/H expression was not detected likely due to the mutation. RNAP expression was also not detected.
<b> Results </b>: DNAP and SSBP protein was successfully expressed. P/H expression was not detected likely due to the mutation. RNAP expression was also not detected.
</p>
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<!-- ==== THIS IS WHERE BIOMINING STARTS ============= -->
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                <h1 id="b_aug_w3"> August - Week 3</h1>               
 +
 +
                  <h3> August 20th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Phosphorylate metal binding DNA sequence from Dunbar lab using Oligonucleotide Phosphorylation protocol prior to ligation. Double digest RsaA with PstI and BglII.
 +
</p><p>
 +
<b> Results </b>: NA.
 +
</p>
 +
                  <h3> August 22th </h3>
 +
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Ligate metal binding DNA sequence into RsaA expression vector.
 +
</p><p>
 +
<b> Results </b>: NA.
 +
</p>
 +
 +
<h1 id="b_aug_w4"> August - Week 4</h1>   
 +
 +
                  <h3> August 27th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Make electrocompetent Caulobacter cells using the electrocompetent cell production protocol.
 +
</p><p>
 +
<b> Results </b>: NA.
 +
</p>
 +
 +
<h1 id="b_sept_w1"> September - Week 1</h1> 
 +
 +
  <h3> September 2nd </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Transform expression vector into electrocompetent Caulobacter cells.
 +
</p><p>
 +
<b> Results </b>: Colonies successfully grew on PYE plate with chloramphenicol.
 +
</p>
 +
 +
                  <h3> September 5th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Pick colonies and inoculate into PYE media
 +
</p><p>
 +
<b> Results </b>: NA.
 +
</p>
 +
 +
<h3> September 6th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Miniprep Caulobacter culture and send the plasmid for sequencing.
 +
</p><p>
 +
<b> Results </b>: NA.
 +
</p>
 +
 +
<h1 id="b_sept_w2"> September - Week 2</h1> 
 +
 +
                  <h3> September 10th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Miniprep Caulobacter culture and send the plasmid for sequencing.
 +
</p><p>
 +
<b> Results </b>: NA.
 +
</p>
 +
 +
<h1 id="b_sept_w3"> September - Week 3</h1>
 +
 +
                  <h3> September 15th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Analyse sequencing results
 +
</p><p>
 +
<b> Results </b>: Sequencing result was positive. The metal binding DNA sequencing was successfully inserted into RsaA expression vector.
 +
</p>
 +
 +
                  <h3> September 18th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: To test effect of fucose on binding affinity to chalcopyrite by doing biopanning assay of fucose knockout and WT Caulobacter.
 +
</p><p>
 +
<b> Results </b>: There was no binding observed in fucose knockout and non-specific binding in WT Caulobacter. See Biomining results – Figure 8.
 +
</p>
 +
 +
<h1 id="b_sept_w4"> September - Week 4</h1>
 +
  <h3> September 20th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Transform recombinant RsaA expression vector into fucose knockout Caulobacter cells.
 +
</p><p>
 +
<b> Results </b>: NA.
 +
</p>
 +
 +
<h3> September 26th </h3>
 +
<p>
 +
<b> Experimenter </b>: Anna Müller, Joe Ho
 +
</p><p>
 +
<b> Aim </b>: Perform biomining assay on fucose knockout Caulobacter with recombinant RsaA expression vector to see if the presence of expression vector improves the binding efficiency. 
 +
</p><p>
 +
<b> Results </b>: See Biomining results – Figure 9.
 +
</p>
 +
 +
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{{:Team:British Columbia/Templates/Footer}}
{{:Team:British Columbia/Templates/Footer}}

Latest revision as of 03:57, 18 October 2014

2014 UBC iGEM

June - Week 3

June 19th

Experimenter : Dan Korvin, Wenchen Zhao, Zeki Ekmekci

Aim : Amplify T7 RNA polymerase gene (RNAP) from T7 bacteriophage genome and terminator from part BBa_B0015 by PCR.

Result : PCR was successful. Target bands were seen on the agarose gel.

June - Week 4

June 23th

Experimenter : Zeki Ekmekci, Wenchen Zhao, Dan Korvin

Aim : Amplify T7 DNA polymerase (DNAP), primase/helicase (P/H), single strand binding protein (SSBP) genes from T7 bacteriophage genome by PCR. Digest terminator with EcoRI (E) and XbaI (X). Digest RNAP with E and SpeI (S).

Results : NA.

June 24th

Experimenter : Ariel Ragetli, Zeki Ekmekci

Aim : Verify DNAP, P/H, SSBP genes on agarose gel. Digest DNAP, P/H, SSBP amplicons with E and S. Ligate each digested T7 gene to digested terminator vector.

Results : DNAP, P/H, SSBP genes successfully amplified, but non-trivial amount of side products (mostly primer-dimer) appeared in DNAP and P/H lanes. Four T7 genes all digested and ligated with terminator.

June 28th

Experimenter : Wenchen Zhao

Aim : Verify the ligation results on agarose gel.

Results : Multiple bands observed (non-trivial amount of side products). Target SSBP+terminator fragment with distinctive amount observed (only one lane with decent result).

June 29th

Experimenter : Jeffrey Pea

Aim : Amplify the target gene/terminator ligated sequence using four different forward primers and same VF reversed primer. Verify the PCR products on a gel.

Results : No significant bands we were looking for observed on the gel.

June 30th

Experimenter : Tudor Lapuste

Aim : Amplify four T7 genes and terminator gene by PCR. Digest four genes using E and S.

Results : SSBP, DNAP and RNAP were amplified successfully. P/H and terminator PCR failed. PCR for P/H and terminator set up.

July - Week 1

July 2nd

Experimenter : Dan Korvin

Aim : Verify P/H + terminator PCR products on agarose gel.

Results : PCR failed for both. More PCR set up for primase/helicase.

July 3rd

Experimenter : Ariel Ragetli, Jeffrey Pea, Zeki Ekmekci

Aim : Verify PCR products for P/H on agarose gel. Amplify terminator by gradient PCR. Transform E.coli cells with terminator-containing plasmid.

Results : Terminator and P/H genes amplified.

July 4th

Experimenter : Dan Korvin

Aim : Digest terminator with E and X. Run PCR products of all four T7 genes on a gel, followed by gel extraction and purification.

Results : NA

July - Week 2

July 6th

Experimenter : Zeki Ekmekci, Wenchen Zhao, Jeffrey Pea

Aim : Double digest all four T7 genes with E and S. Run digested T7 genes and terminator on a gel, followed by gel extraction and purification. Ligate T7 genes with terminator. Transform all four constructs to E.coli

Results : NA

July - Week 3

July 10th

Experimenter : Wenchen Zhao, Zeki Ekmekci

Aim : Verify constructs by colony PCR.

Results : Colony PCR failed. No bands were observed.

July 17th

Experimenter : Jeffrey Pea

Aim : Double digest terminator plasmid with E and X. Ligate digested T7 genes to terminator. Transformed four constructs to E.coli

Results : NA

July - Week 4

July 20th

Experimenter : Tudor Lapuste

Aim : Verify constructs by colony PCR.

Results : SSBP and RNAP constructs (gene + terminator) confirmed.

July 24th

Experimenter : Ariel Ragetli, Jeffrey Pea

Aim : Re-amplify DNAP and P/H by PCR followed by gel check. Ligate two digested amplicons to digested terminator. Transform two constructs into E.coli.

Results : DNAP and P/H were successfully amplified.

August - Week 1

August 1st

Experimenter : Wenchen Zhao, Dan Korvin, Zeki Ekmekci

Aim : Verify DNAP and P/H constructs by colony PCR. Digest SSBP and RNAP constructs with E and S. Digest the plasmid containing Pbad promoter and RBS with E and X. Transform the ligation products to E.coli.

Results : Colony PCR failed.

August 6th

Experimenter : Wenchen Zhao, Jeffrey Pea, Zeki Ekmekci

Aim : Verify DNAP and P/H constructs (gene+terminator), SSBP and RNAP constructs (gene+terminator+RBS+promoter) using colony PCR.

Results : P/H and DNAP constructs confirmed. Bands with correct size were found on the gel after colony PCR of SSBP and RNAP constructs.

August - Week 2

August 12th

Experimenter : Wenchen Zhao, Zeki Ekmekci

Aim : Prepare SSBP and RNAP miniprep products and send them for sequencing. Double digest P/H and DNAP constructs with E and S. Ligate digested constructs into vector digested with E and X. Transform ligation products into E.coli.

Results : NA.

August - Week 3

August 19th

Experimenter : Ariel Ragetli, Zeki Ekmekci

Aim : Verify P/H and DNAP constructs using colony PCR. Analyze sequencing results of SSBP and RNAP complete constructs.

Results : Sequencing results are negative – no Pbad promoter/RBS found in SSBP and RNAP constructs. Colony PCR result was also negative.

September - Week 1

September 4th

Experimenter : Dan Korvin, Wenchen Zhao

Aim : Double digest DNAP, RNAP and SSBP constructs with X and PstI (P). Ligate three digested genes into RBS + Pbad Promoter plasmid digested with S and P. Miniprep P/H construct.

Results : NA.

September 5th

Experimenter : Wenchen Zhao

Aim : Transform all three constructs into E.coli. Site-directed mutagenize P/H construct and transform into E.coli.

Results : NA.

September 6th

Experimenter : Anna Müller

Aim : Verify DNAP, RNAP and SSBP constructs by colony PCR.

Results : Bands with correct size observed, need to send to sequencing for further verification.

September - Week 2

September 8th

Experimenter : Anna Müller

Aim : Miniprep all cultures including SDM P/H construct with terminator and complete DNAP, RNAP and SSBP constructs

Results : NA

September 9th

Experimenter : Wenchen Zhao, Zeki Ekmekci

Aim : Send DNAP, RNAP, SSBP constructs for sequencing. Double digest P/H constructs with X and P, followed by gel check. Double digest RNAP with E and X.

Results : Gel check result was negative. No target band was observed. P/H was not mutagenized.

September 10th

Experimenter : Ariel Ragetli, Jeffrey Pea

Aim : Double digest SSBP with E and S. Double digest RNAP with E and X, followed by gel check and extraction. Ligate digested SSBP into digested RNAP vector. Transform ligation product into E.coli.

Results : NA.

September - Week 3

September 15th

Experimenter : Wenchen Zhao

Aim : Colony PCR SSBP + RNAP construct. Repeat SDM of P/H construct.

Results : Colony PCR failed.

September 16th

Experimenter : Wenchen Zhao

Aim : Re-run colony PCR of SSBP and RNAP construct.

Results : Colony PCR result was positive.

September 17th

Experimenter : Wenchen Zhao

Aim : Miniprep SSBP + RNAP construct, double digest with E and S. Double digest DNAP with E and X, followed by gel verification and extraction. Ligate digested DNAP with digested SSBP + RNAP construct. Transform SDM P/H plasmid.

Results : NA.

September 18th

Experimenter : Wenchen Zhao

Aim : Colony PCR SDM P/H. Double digest P/H with X and P.

Results : Colony PCR failed.

September 19th

Experimenter : Wenchen Zhao

Aim : Repeat colony PCR of SDM P/H. Miniprep DNAP, RNAP for sequencing.

Results : Colony PCR results were positive.

September - Week 4

September 21st

Experimenter : Zeki Ekmekci

Aim : Double digest 3-gene assembly with E and P. Double digest SDM P/H with X and P. Ligate digested P/H to digested RBS plasmid. Transform ligation product to E.coli.

Results : NA.

September 23rd

Experimenter : Wenchen Zhao

Aim : Colony PCR P/H construct followed by gel check.

Results : Colony PCR result was positive.

September 24th

Experimenter : Wenchen Zhao

Aim : Analyze sequencing results for DNAP, SSBP and RNAP constructs. Miniprep SDM P/H and send it for sequencing.

Results : Sequencing results were positive. DNAP, SSBP and RNAP constructs were successfully assembled.

October - Week 1

October 1st

Experimenter : Wenchen Zhao

Aim : Analyze sequencing results for P/H construct. Digest P/H with E and S and digest 3-gene assembly with E and X. Ligate double digest P/H into double digested 3-gene vector. Transform the ligation product to E.coli.

Results : Sequencing result was negative. Pbad romoter/RBS was not present in the sequence. But XbaI restriction site was successfully mutated.

October 2nd

Experimenter : Wenchen Zhao

Aim : Double digest SDM P/H with X and P. Double digest promoter/RBS plasmid with S and P. Ligate digested P/H into digested promoter/RBS plasmid. Transform the ligation product into E.coli.

Results : NA

October 3rd

Experimenter : Wenchen Zhao

Aim : Verify P/H constructs by colony PCR.

Results : Colony PCR result was positive. Target band was observed on the gel.

October - Week 2

October 4th

Experimenter : Ariel Ragetli

Aim : Verify P/H constructs by colony PCR.

Results : Colony PCR result was positive. Target band was observed on the gel.

October 7th

Experimenter : Jeffrey Pea

Aim : Miniprep P/H plasmid and send it for sequencing.

Results : NA

October 8th

Experimenter : Jeffrey Pea

Aim : Double digest P/H with E and X. Double digest 3-gene assembly with E and S. Ligate digested P/H into digested 3-gene assembly. Transform the ligation product to E.coli.

Results : NA

October 9th

Experimenter : Jeffrey Pea

Aim : Verify 4-gene assembly by colony PCR followed by PCR.

Results : Colony PCR result was positive. 4-gene construct was assembled.

October 10th

Experimenter : Wenchen Zhao

Aim : Analyze sequencing result for P/H plasmid.

Results : Sequencing result was negative. There was a ~200bp deletion in the middle of P/H gene. Also there was a 2bp deletion at the beginning of the gene, causing a frameshift mutation

October - Week 3

October 14th

Experimenter : Wenchen Zhao

Aim : Verify protein expression of 4-gene assembly on SDS-PAGE.

Results : DNAP and SSBP protein was successfully expressed. P/H expression was not detected likely due to the mutation. RNAP expression was also not detected.

August - Week 3

August 20th

Experimenter : Anna Müller, Joe Ho

Aim : Phosphorylate metal binding DNA sequence from Dunbar lab using Oligonucleotide Phosphorylation protocol prior to ligation. Double digest RsaA with PstI and BglII.

Results : NA.

August 22th

Experimenter : Anna Müller, Joe Ho

Aim : Ligate metal binding DNA sequence into RsaA expression vector.

Results : NA.

August - Week 4

August 27th

Experimenter : Anna Müller, Joe Ho

Aim : Make electrocompetent Caulobacter cells using the electrocompetent cell production protocol.

Results : NA.

September - Week 1

September 2nd

Experimenter : Anna Müller, Joe Ho

Aim : Transform expression vector into electrocompetent Caulobacter cells.

Results : Colonies successfully grew on PYE plate with chloramphenicol.

September 5th

Experimenter : Anna Müller, Joe Ho

Aim : Pick colonies and inoculate into PYE media

Results : NA.

September 6th

Experimenter : Anna Müller, Joe Ho

Aim : Miniprep Caulobacter culture and send the plasmid for sequencing.

Results : NA.

September - Week 2

September 10th

Experimenter : Anna Müller, Joe Ho

Aim : Miniprep Caulobacter culture and send the plasmid for sequencing.

Results : NA.

September - Week 3

September 15th

Experimenter : Anna Müller, Joe Ho

Aim : Analyse sequencing results

Results : Sequencing result was positive. The metal binding DNA sequencing was successfully inserted into RsaA expression vector.

September 18th

Experimenter : Anna Müller, Joe Ho

Aim : To test effect of fucose on binding affinity to chalcopyrite by doing biopanning assay of fucose knockout and WT Caulobacter.

Results : There was no binding observed in fucose knockout and non-specific binding in WT Caulobacter. See Biomining results – Figure 8.

September - Week 4

September 20th

Experimenter : Anna Müller, Joe Ho

Aim : Transform recombinant RsaA expression vector into fucose knockout Caulobacter cells.

Results : NA.

September 26th

Experimenter : Anna Müller, Joe Ho

Aim : Perform biomining assay on fucose knockout Caulobacter with recombinant RsaA expression vector to see if the presence of expression vector improves the binding efficiency.

Results : See Biomining results – Figure 9.

http://2014.igem.org/Team:British_Columbia/Notebook/Labbook"