Team:Cambridge/Notebook/10

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Latest revision as of 21:25, 26 October 2010

Week 10: Monday 13th - Sunday 19th September

Monday

To our pleasant surprise our plates of P. pyralis in pSB1C3, L. cruciata in pSB1C3 and L. cruciata with pBAD in SB1C3 had all grown successfully over the weekend. Only P. pyralis with pBAD had not grown, which Hannah and Emily (who was back from holiday) repeated that experiment today.

In the meantime, Bill investigated flights to America and Theo wrote a draft abstract which the team discussed over coffee. The thioesterase plates had also grown over the weekend as well, which was very exciting! Ben set up a plate reader experiment going overnight to test whether L. cruciata luciferase worked and Theo took very pretty pictures of light bulbs glowing with bacteria!

In trying to be organised, we've also grown up broths of the original DNA2.0 plasmids and the products of the plates to make glycerol stocks tomorrow. Ben put on a plate reader experiment in the evening while Theo made overnight cultures that could be sent for sequencing.

Tuesday

We sent a number of sequences for sequencing, courtesty of Source BioSciences. Unfortunately in our hurry to meet the deadline we broke a pipette, and we missed it anyway. But the analysis of the plate reader experiment showed us that the Luciola cruciata luciferase was indeed working. Hannah and Emily performed standard Biobrick assembly to try to ligate the pBAD promoter onto the EPIC mutant of Photinus pyralis luciferase and LRE operon, which failed before. We got very excited about blog posts by our very own [http://labrat.fieldofscience.com/2010/09/bacterial-lightbulbs.html lab rat] and also [http://scienceblogs.com/oscillator/2010/09/bacterial_lightbulb.php Oscillator]. Theo plated out Luciola cruciata on an arabinose containing plate in order to make a red bulb to add to our blue.

Wednesday

The ligation had failed so Emily and Hannah redid their experiment. Meanwhile, Ben attempted to use site-specific PCR mutagenesis followed by Gibson assembly to mutate the red mutant of Luciola cruciata luciferase back to wild type. This is a first step needed so that we can mutate it into a number of other colours.

Thursday

Since Hannah and Emily's experiment did not seem to have produced a viable ligation, Theo tried this yet again. But over the course of the day we noticed small colonies growing on a plate from the first ligation, which was an encouraging result. Ben retried the Gibson assembly and the team rehearsed a Gibson Assembly song Theo had written the previous evening. Ben arranged it for ukelele!

Friday

Today we got the robot in Jim's lab to do some work for us, it drew iGEM in Pixels on a 96 well plate! We hope to get this working with all the firefly colours eventually, and hopefully to mix them together. We also tidied the lab, and continued construction of a bacterial bubble lamp.

Saturday

Theo and Bill came into the lab. Bill performed a number of Gibson assemblies attempting to separate LRE and luciferase BioBricks. Theo tried continuing Ben's work on site directed mutagenesis of Luciola cruciata luciferase back to wild-type.

@@ Line 1: / Line 1: @@
 <html>
-<div align="center">Week 9: Monday 6th - Sunday 12th September</div>
+<div align="center">Week 10: Monday 13th - Sunday 19th September</div>
 </html>
 {{Team:Cambridge/Templates/Day|Day=Monday}}
-{{:Team:Cambridge/Templates/rightpic|src=Cambridge-DNA20.jpg}}
+{{:Team:Cambridge/Templates/rightpic|src=Cambridge-bluebulb.jpg}}
-This morning was very exciting because both our orders from DNA2.0 had arrived.  These were the firefly operons, both containing both a luciferase and a luciferin regenerating enzyme.  However we became incredibly paranoid, since the two tiny discs of filter paper were worth a great deal!
+To our pleasant surprise our plates of P. pyralis in pSB1C3, L. cruciata in pSB1C3 and L. cruciata with pBAD in SB1C3 had all grown successfully over the weekend. Only P. pyralis with pBAD had not grown, which Hannah and Emily (who was back from holiday) repeated that experiment today.
-DNA 2.0 had supplied a protocol for extraction but we wanted to OK it with our advisors first and there was no-one in the lab, so we regretfully decided to leave extraction and transformation for Tuesday.
+In the meantime, Bill investigated flights to America and Theo wrote a draft abstract which the team discussed over coffee. The thioesterase plates had also grown over the weekend as well, which was very exciting! Ben set up a plate reader experiment going overnight to test whether L. cruciata luciferase worked and Theo took very pretty pictures of light bulbs glowing with bacteria!
+In trying to be organised, we've also grown up broths of the original DNA2.0 plasmids and the products of the plates to make glycerol stocks tomorrow. Ben put on a plate reader experiment in the evening while Theo made overnight cultures that could be sent for sequencing.
-However we did see interesting results in that G28, an attempt Will had made to Gibson assemble the Vibrio fischeri lux operon into E. coli had glowed in the plate reader.  Therefore Theo made up plates containing arabinose in LB with chloramphenicol and plated some out.
 {{Team:Cambridge/Templates/Day|Day=Tuesday}}
-{{:Team:Cambridge/Templates/rightpic|src=Cambridge-G28First.jpg‎}}
+{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Tue.jpg‎}}
-G28 had indeed been induced and glowed, see the pic on right - the first photograph of one of our finished BioBricks emitting light (since nothing else has been in pSB1C3).  We still need to sequence it to show it is definitely correct.
+We sent a number of sequences for sequencing, courtesty of Source BioSciences.  Unfortunately in our hurry to meet the deadline we broke a pipette, and we missed it anyway. But the analysis of the plate reader experiment showed us that the Luciola cruciata luciferase was indeed working.  Hannah and Emily performed standard Biobrick assembly to try to ligate the pBAD promoter onto the EPIC mutant of Photinus pyralis luciferase and LRE operon, which failed before.  We got very excited about blog posts by our very own [http://labrat.fieldofscience.com/2010/09/bacterial-lightbulbs.html lab rat] and also [http://scienceblogs.com/oscillator/2010/09/bacterial_lightbulb.php Oscillator].  Theo plated out Luciola cruciata on an arabinose containing plate in order to make a red bulb to add to our blue.
-Today we also started a number of standard Biobrick assembly operations.  We wanted to move the registry luciferase under a TetR promoter into pSB1C3 and also to add a fluorescent reporter to this operon, as well as G28.
-As such we performed a restriction digest, ran gels and performed gel extraction. We put this DNA in the freezer and turned our attention to our order from DNA 2.0.
-This was extracted from filter paper adding Tris-HCl and centrifuging.  Then we transformed competent cells, we performed 4 transformations for each construct just in case, we plated them all out on kanamycin plates to select for the DNA 2.0 in house vector [https://www.dna20.com/index.php?pageID=278 pJ207].
 {{Team:Cambridge/Templates/Day|Day=Wednesday}}
-{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Box.jpg‎}}
+{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Wed.jpg‎}}
-To our immense relief the DNA 2.0 transformations had indeed grown up!  We prepared overnight cultures for BioBricking.
+The ligation had failed so Emily and Hannah redid their experiment.  Meanwhile, Ben attempted to use site-specific PCR mutagenesis followed by Gibson assembly to mutate the red mutant of Luciola cruciata luciferase back to wild type.  This is a first step needed so that we can mutate it into a number of other colours.
+{{Team:Cambridge/Templates/Day|Day=Thursday}}
+{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Uke.jpg‎}}
+Since Hannah and Emily's experiment did not seem to have produced a viable ligation, Theo tried this yet again.  But over the course of the day we noticed small colonies growing on a plate from the first ligation, which was an encouraging result.  Ben retried the Gibson assembly and the team rehearsed a Gibson Assembly song Theo had written the previous evening.  Ben arranged it for ukelele!
+{{Team:Cambridge/Templates/Day|Day=Friday}}
+{{:Team:Cambridge/Templates/rightpic|src=Cambridge-iGEMpixels.jpg‎}}
+Today we got the robot in Jim's lab to do some work for us, it drew iGEM in Pixels on a 96 well plate!  We hope to get this working with all the firefly colours eventually, and hopefully to mix them together.
+We also tidied the lab, and continued construction of a bacterial bubble lamp.
-Also Paul continued in his attempts to extract the exisiting E. coli thioesterase gene from genomic DNA for BioBricking.  Unfortunately the DNA was so faint that it was best visualised by putting a cardboard box over the transilluminator.
-Theo ligated the fluorescent proteins onto the two operons, continuing Tuedays experiment and plated these out.
-{{Team:Cambridge/Templates/Day|Day=Thursday}}
-==Friday==
 ==Saturday==
+Theo and Bill came into the lab.  Bill performed a number of Gibson assemblies attempting to separate LRE and luciferase BioBricks.  Theo tried continuing Ben's work on site directed mutagenesis of Luciola cruciata luciferase back to wild-type.
 ==Sunday==