iGEM 2014 — Team UGA-Georgia

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# Team:UGA-Georgia/Notebook

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| |  | | ![](https://static.igem.org/mediawiki/2014/f/fa/Bannerugafinal.png)  [HOME](https://2014.igem.org/Team%3AUGA-Georgia) | **PROJECT** [Overview](%E2%80%9Chttps%3A//2014.igem.org/Team%3AUGA-Georgia/Overview%22) [Geraniol](https://2014.igem.org/Team%3AUGA-Georgia/Geraniol) [Modeling](https://2014.igem.org/Team%3AUGA-Georgia/Modeling) [RBS Library](https://2014.igem.org/Team%3AUGA-Georgia/RBS) [Parts](https://2014.igem.org/Team%3AUGA-Georgia/Parts) | **WET LAB** [Safety](https://2014.igem.org/Team%3AUGA-Georgia/Safety) [Notebook](https://2014.igem.org/Team%3AUGA-Georgia/Notebook) [Protocols](https://2014.igem.org/Team%3AUGA-Georgia/Protocols) | **HUMAN PRACTICES** [Outreach](https://2014.igem.org/Team%3AUGA-Georgia/Outreach) [Seminars](https://2014.igem.org/Team%3AUGA-Georgia/Seminars) | **TEAM** [Official Team Profile](https://igem.org/Team.cgi?id=1383) [Members](https://2014.igem.org/Team%3AUGA-Georgia/Team) [Attributions](https://2014.igem.org/Team%3AUGA-Georgia/Attributions) | [![](https://static.igem.org/mediawiki/2014/d/d2/UGA-iGEM_Logo.jpg)](https://2014.igem.org/Main_Page) | | | ||  | | |
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| Notebook | | |
| February  Week 1- Introduction to anaerobic facilities for new members  Week 3- Making of NaS solution. - Making of formate media.  Week 4- Creation of transformation buffer (TB). - First geraniol extraction efficiency test with balch tubes.     March  Week 1- Creation of geraniol standards for gas chromatography (GC) analysis/calibration through serial dilutions.  Week 3- Creation of more formate media. - Creation of general salts solution. - Creation of glycylglycine buffer.  * All of these solution protocols can be found on our [protocol](https://2014.igem.org/Team%3AUGA-Georgia/Protocols) page.      April  Week 1- Learning about ribosome binding site library (RBS) and our metabolic Optflux model.  Week 2- Made agar plates - Creation of fresh wild type (WT) stocks.  Week 3- Created diluted puromycin     May  Week 4- Inoculation and revival of frozen stocks.     June  Week 1- Determined the optical density (OD) of the previously revived cultures.  Week 2- Restriction digestion, purification, and ligation of pMEV4 and PCR products (C1 and C2). - Transformation by heat shock, plated the cells, made an agarose gel and ran the products on it. - Picked colonies from the previous day of plating. - Plasmid extraction, digestion, gel electrophoresis, and verification. - [E. Coli Lab] Extracted pMEV4 from bacteria. - [E.Coli Lab] PRC for mCherry with 11 mutated RBS sites and 1 native RBS. - [E. Coli Lab] Vector pMEV4 was digested with Spe1, Pst1 and buffer 2. Gel Extraction, Ligation and Heat Shock Transformation were also performed. - [E. Coli Lab] Colonies were successfully formed from 1,3,4,8, and 11; PCR was redone for 2,5,6,7,10, and 12. - [E. Coli Lab] Created new LB plates with amplicillin and LB Broth. - [E. Coli Lab] Prepared competent cells for transformation - [E. Coli Lab] Obtained PCR product samples 2,5,6,7,10 and 12, and redid vector digestion, gel extraction, ligation, and heat shock transformation. - [E. Coli Lab] Sample 5,6,7 and 10 were unsuccessfully transformed. Redid vector digestion, gel extraction, ligation, and heat shock transformation.  Week 3- Revival of pAW50-mCherry frozen stock. - Revival of S0001 (WT) frozen stock. - [E. Coli Lab] Inoculation, plasmid extraction, digestion and screening were performed on 1, 3,4,5,8,9,10,11 and 12. - [E. Coli Lab] Screening [Part I](https://static.igem.org/mediawiki/2014/7/7f/Screening1-12.png), [Part II](https://2014.igem.org/File%3APart2screening.png) for 1,3,4,5,8,9,10,11 and 12. Verification was done by using KpnI and NcoI ( if positive:2750 and 2414; if negative: 2414 and 1986; control: pMEV4). - [E. Coli Lab] Cloning for 2,6, and 7 in progress  Week 4- Prepared the revived cultures for fluorescence microscopy. - Fluorescence microscopy training. - Anaerobic transformation of RBS libraries 1-12 into  *M. maripaludis.* - Puromycin enrichment of transformants. - Made glycerol stocks of original transformants. - Fluorescent microscopy of transformants/pAW50-mCherry/S0001. - [E. Coli Lab] Plasmid extraction was done for 1,3,4,5,8,9,10,11 and 12. Also made 2 permanent stocks for each of them. - [E. Coli Lab] Plasmid extraction, inoculation, and screening were performed on 2, 6 and 7. Also made 2 permanent stocks for each of them     July  Week 1- Took ODs of the native RBS and library 1 and 2 (12; L-1; L-2 respectively). - Plated the 12, L-1, and L-2 cultures. - Dispensed media to test tubes. - Picked colonies form the plates.  Week 2- ODs from last weeks picked colonies were taken. - Made frozen stocks of RBS colonies, - Sub-cultured parent strains. - Researched primary literature on mCherry maturation. - Began developing a protocol for oxygen maturation of mCherry.  Week 3- Made frozen stocks of RBS colonies and sub-cultured parent strains. - Re-subcultured and revive pAW50-mcherry. - Subcultured the parent strains of WT and native RBS (12) into triplicates. The triplicates were wrapped in foil to prevent any photobleaching. - The pAW50-mcherry came up and then all samples were taken to a plate reader to perform fluorescence tests. - [E. Coli Lab] the primers for the negative and positive control group were received; ran PCR on the negative and control groups in order to create a large amount of the DNA. - [E. Coli Lab] PCR amplification for mCherry gene with the negative and positive control. - [E. Coli Lab] Solid media was made. - [E. Coli Lab] [PCR](https://static.igem.org/mediawiki/2014/a/a4/13-15PCRgel.png) was performed on 13, 14 and 15; vector digestion was also performed with Spe1, Pst1 and buffer2. - [E. Coli Lab] Gel electrophoresis was done to confirm [digestion](https://static.igem.org/mediawiki/2014/4/4b/13-15Diggel.png) of 13-15.  Week 5- Filled out and submitted safety form. - The geraniol extraction efficiency experiment was performed.  * Left the organic phase to dry over night and be resuspended to test for voltility.  - Track selection.     August  Week 2- Geraniol synthase containing cells (GS) were inoculated into triplicates of varying concentrations of puromycin.  * The previous geraniol extraction methods were repeated with these new GS samples.   Week 3- Geraniol standards were made to be test on GC/MS. - We did an extraction efficiency test between batch tube vs. separatory funnel. - Testing of geraniol on the GC/MS was done.  Week 4- Cells containing our pMEV4 plasmid with native RBS were inoculated into 5mL of rezasurin free (RF) media with varying levels of puromycin concentration. - ODs were taken of the samples and they were placed into darkness.  Week 5- The cells were prepared for the plate reader as per the first plate reader preparation protocol. - The plate reader results were in conclusion and a new protocol was required.     September  Week 2- A frozen stock of pAW50-mCherry was revived in 5mL of RF media. - Room temperature stocks of 3 colonies of 12 were inoculated into 5mL of RF media.  * All of these will act as parent strains that will then be inoculated and grown in varying conditions to test for the best condition for generating mCherry.   Week 3- The parent strains were inoculated and grown in the varying conditions.  * 37C vs. 30C * 100mL vs. 5mL media  - Some of each of the parent strains were also PCR tested to confirm the presence of the mCherry gene.  * The PCR showed that the mCherry gene was indeed present.   Week 5- ODs of the pAW50-mCherry and the 3 colonies of 12 that were grown in varying conditions were taken and they were all above OD=0.6. - The samples were prepared for overnight oxygen exposure as per the protocol. - The samples were taken to the plate reader to measure fluorescence.- * Fluorescence was measured for all the 12 samples in each condition.- Since this pre-study gave us consistent, positive values for fluorescence, we were able to now refine the oxygen exposure protocol and apply it to characterization of our parts.     * This detailed protocol can be found in our [protocol](https://2014.igem.org/Team%3AUGA-Georgia/Protocols) section.- Revival of frozen stocks of cells containing plasmids with our theoretical "perfect" RBS (14), cells containing our theoretical "negative" RBS (15), and each of the 3 colonies of 12. Also, revival of WT with no plasmid were revived from a room temperature stock (negative control).     - [E. Coli Lab] PCR Verification of 12C1, 12C2 and 12C3 was done with F12 as forward primer and R as reverse primer.             October  Week 1- The revived parent strains of 14, 15, WT, and 12 C-1,2,3 were inoculated into varying volumes, and in triplicates, to test for a linear relationship of mCherry production to volume of culture which would be determined after the samples were taken to the plate reader.     * The triplicates were grown in 5mL, 25mL, and one single sample each of 12 C-1, 14, and 15 were grown into 100mL as a reference.- [E. Coli Lab] Cloning for BioBrick (Genes: BBa\_K1383000{Native RBS; F12 with F12 and R primers}, BBa\_K1383001{Theoretical Best RBS; F14 with F14 and R primers}, and BBa\_K1383002{Theoretical Worst RBS; F15 with F15 and R primers}; Vector: pSB1C3); PCR [result](https://static.igem.org/mediawiki/2014/0/08/Biobrickpcr.png)  was observed by using gel electrophoresis and UV light.     - [E. Coli Lab] Plasmid extraction, purification, and  [screening](https://static.igem.org/mediawiki/2014/a/a4/Biobrickscreening10-03-2014.JPG)  were performed on F12A, F12B, F12C, F14A, F14B, F14C, F15A, F15B, and F15C.      Week 2- The oxygen exposure [protocol](https://2014.igem.org/Team%3AUGA-Georgia/Protocols) was performed on the triplicates and the results of the experiment can be found on our [RBS Library](https://2014.igem.org/Team%3AUGA-Georgia/RBS) page. |  |  |

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