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Team:Nevada/Notebook/Weeks1316
From 2011.igem.org
(→Week 14 - August 29th- September 4th) |
(→Week 14 - August 29th- September 4th) |
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<u>Pyruvate Decarboxylase & Alcohol Dehydrogenase</u><br><br> | <u>Pyruvate Decarboxylase & Alcohol Dehydrogenase</u><br><br> | ||
| - | JC: To again test PDC and ADH activity, | + | JC: To again test PDC and ADH activity, s70/PDC/ADH was transformed into NEB High Expression Iq cells. These cells were plated on the aldehyde detection plates and demonstrated a more intense pink colony than the NEB B10 cells. |
<br><br> | <br><br> | ||
Revision as of 17:13, 23 September 2011
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Calender Weeks 13-16
To edit on a specific week. Click on the edit button corresponding to the week.
Contents |
Week 13 - August 22nd-28th
E. Coli
Trc Promoter
LT:The protocol from week #11 was repeated but the transformation reaction was plated onto LB-CHL plates for proper selection. Results: The BTE digest was successful as confirmed by the gel. The trc promoter digest had the same inconclusive results as seen preciously. Colonies successfully grew from the ligation and transformation.
Pyruvate Decarboxylase & Alcohol Dehydrogenase
JC: To test viability of PDC, found information regarding Aldehyde detection plates. Since PDC converts Pyruvate to Acetaldehyde, these plates could be a quick way to test for the presence of aldehydes in our constructs. Growing s70/PDC/ADH in NEB B10 cells yielded bright pink colonies (aldehydes present).
Bay Laurel Thioesterase
MT: Five cultures of BTE coding region in the Topo vector (pUC57) were grown, and minipreped. The BTE samples and pSB1C3 were digested with Xba I and Pst I and ligated. Ligation was transformed using NEB Beta cells. Colonies were selected to culture overnight and minipreped, digested to check for correct band size.
A Free Fatty Acid Assay was done on BTE with the promoter sigma 70. A sample was also ran through a Gas chromatographer to check the length of hydrocabons we are producing. Results are in the assay section.
BTE with sigma 70 promoter and pSB1C3 were digested with EcoRI and PstI and ligated, transformed, and cultured.
Cyano
Comment Here
Enzymology
CL:This week we used the GC to analyze the fatty acids being produced by one of the BTE IQ cell samples (BTE13). The fatty acids were extracted from cell supernatent using hexane and tested with a carbon disulfide solvent. We used FAME standards ranging from C:8-C:19 as well as a C:19 internal standard for comparison. Results proved positive as we were looking for C:12 FA's and there were C:8, C:10 and C:12 FA's present in the sample. We suspect that the small chains could possibly be the larger C:12 chain being metabolized and we plan to further analyzed this data. This was just a test or a pre-run and we did not use a negative control or test very many samples. We plan to do a definite run next week on four new BTE samples as well as negative controls of wild-type IQ cells and a known concentration of a C:17 internal standard will be used for quantification. Also this week we performed our third ethanol assay using the BioSystems Ethanol assay kit on 4 of the ADH/PDC IQ cell cultures. We retrieved positive results for one of the samples (ACH/PDC17) with an ethanol production of 3.4mM. We are planning to perform the ADH activity assay on this culture and four new samples next week.
Media
Comment Here
Week 14 - August 29th- September 4th
E. Coli
Trc Promoter
LT:Colonies #1-15 were selected from the BTE/trc promoter transformation, inoculated in TB-CHL, and minipreped. Colonies #16-45 were selected and used for colony PCR using VF and VR primers. Half the PCR products were loaded onto a 0.7% gel to check the products. Results: There is no DNA present on the gel. It appears that a mistake was made during preparation of the PCR reaction. Only the standards were visible.
Pyruvate Decarboxylase & Alcohol Dehydrogenase
JC: To again test PDC and ADH activity, s70/PDC/ADH was transformed into NEB High Expression Iq cells. These cells were plated on the aldehyde detection plates and demonstrated a more intense pink colony than the NEB B10 cells.
Bay Laurel Thioesterase
MT: The cultures of the transformation of BTE with sigma 70 promoter in pSB1C3 were minipreped, digested with EcoRI and PstI, and checked on a 1% gel. Four of the five had full digestion. Sample #4 was sent for sequencing.
Cyano
Comment Here
Enzymology
Comment Here
Media
Comment Here
Week 15 - September 5th-11th
E. Coli
Comment Here
Cyano
Comment Here
Enzymology
Comment Here
Media
Comment Here
Week 16 - September 12th-18th
E. Coli
Bay Laurel Thioesterase
MT: The sequencing results of sigma 70/ BTE in pSB1C3 showed a succseful clone and is ready for submission to iGEM.
A Free Fatty Acid Assay was done on the BTE with sigma 70 promter in Iq cells. The samples were also ran through a Gas Chromotagrapher to determine concentration of C-12 fatty acids. ( For more specific results please look in Enymology section.)
Cyano
Comment Here
Enzymology
Comment Here
Media
Comment Here
Week 17 - September 19th-25th
E. Coli
Comment Here
Cyano
Comment Here
Enzymology
Comment Here
Media
Comment Here
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/* Wiki Hacks - START */
/* Author: Pieter van Boheemen */
/* Team: TU Delft */
/* Thanks guys - Bill Collins */
/* +1 - Douglas Watson */
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