Team:Cambridge/Notebook/9

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Monday

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!

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.

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.

Tuesday

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. 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 pJ207.

Wednesday

To our immense relief the DNA 2.0 transformations had indeed grown up! We prepared overnight cultures for BioBricking.

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.

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-We came in in the morning and viewed the results from the plate reader experiment that had been running overnight. The bacterial lux results were disappointing.  Our positive control was emitting (blue) light.  (See photo on the right), but the other cells which had been incubated with arabinose in the hope of inducing them had not emitted light.
-But the most interesting results came from the cells expressing firefly luciferase ([http://partsregistry.org/Part:BBa_I712019 BBa_I712019]) under a consitutive promoter / RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J13002 BBa_J13002]. These showed low light output for 13 hours and then a rapid increase in luminescence, to a level which was sustained until the evening. We left the cells in the reader overnight to continue reading.
 ==Friday==
-We got results from sequencing of what we hoped to be our first Biobrick.  It was not what we had hoped for, which was dissapointing.  Additionally, Theo miniprepped a lux operon assembled using Gibson techniques and found it was the wrong size, after restriction enzyme cutting and running on a gel.
-But on a positive note synthesis from DNA 2.0 arrived, and preliminary results using CHBT combined with D-cysteine showed significant light output implying that functional LRE should allow recycling of oxyluciferin.
 ==Saturday==
 ==Sunday==
-Theo made a [https://2010.igem.org/Team:Cambridge/Tools/GenBank tool] to allow export of BioBricks with annotations.
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Team:Cambridge/Notebook/9

From 2010.igem.org

Revision as of 19:26, 8 September 2010

Contents

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday