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We are sequencing the genome of Strain E-15 EG3 so that we can see what changes are present between the MG1655 and Strain E-15 EG3. Once sequenced, we will be able to compare the sequences between each strain which would show single nucleotide polymorphisms (SNPs), as well as any deletions or insertions. From this, we will be able to apply the knowledge to a hybrid approach with the rational engineering.  

Using the transposase, Tn5, we have constructed an Illumina compatible library that we plan to submit to our Sequencing Core Facilities. Unfortunately, the sequencing run on the MiSeq takes around one week and we were not able to get sequence data before the competition.

<p>Re-streaks</p>

After receiving Strain E-15 EG3, the stab culture was re-streaked on ethylene glycol (EG) agar for three generations to ensure that the strain retained the two functional enzymes key for ethylene glycol utilization after thirty years of storage. Colonies became defined on the EG plates every three to four days, and were immediately re-streaked onto a new EG plate. With this selection pressure to choose the fastest developing colony, our hope is to isolate an evolved version of Strain E-15 EG3, that is capable of replicating on EG at a faster pace.

<br>We are comparing the most recent re-streak (re-streak #12) with the earliest re-streak (re-streak #3) to test if constantly selecting Strain E-15 EG3 improves the overall growth rate of the strain. <br><br>

<p>Liquid Cultures</p>

We made liquid cultures of the re-streaked Strain E-15 EG3 and re-passaged the strain in new ethylene glycol media for twenty-five generation cycles each time the tubes reached an OD 600 of 0.2. By repeating this process, we were constantly selecting the fastest growing cells on ethylene glycol. We hoped to create a population whose growth rate was even greater than the original Strain E-15 EG3. (Click <a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol#Directed">here</a> for data relating to the repassaging.) Since the liquid culture contains a mixed population of EG utilizing cells, it is important to first plate the cells and select for the fastest developing colonies. After observing the presence of colonies, the first fifty-five colonies were selected, tested for rate of growth in EG media, and compared to the standard, Strain E-15 EG3 from re-streak #3. <br>

<br><center><img src="https://static.igem.org/mediawiki/2012/3/3c/ODcuvettereading.JPG" align="left"><img src="https://static.igem.org/mediawiki/2012/8/85/Clearflaskwithtip.JPG" align="right"></center><br><br><br><br><br><br><br><br><br><br><br><br>

<p>EMS mutagenesis</p>

Ethyl methanesulfonate (EMS) is a liquid mutagen that favors a G/C to A/T transition. We are employing the EMS to both the MG1655 and E-15 EG3 liquid cultures in different concentrations (28 μL, 56 μL, 84 μL to 4 mL of cells) for different amounts of time (0 minute, 15 minutes, 30 minutes, and 45 minutes) [3]. However, we are not focusing on a specific gene with a high G/C content, so the EMS just serves the purpose of adding variation to the population. We wanted to compare the effects of EMS and whether mutagenesis introduced any variation in the utilization of ethylene glycol at different rates. To set up the comparison across plates for relative growth rates, a set of Strain E-15 EG3 standards were included in each plate. We expect that the EMS caused some variation in each of the populations. However, they are probably minute variations, and are likely to be similar in efficiency. <br><br><img src="https://static.igem.org/mediawiki/2012/6/67/EMSatArcadia1.JPG" align="left"><img src="https://static.igem.org/mediawiki/2012/4/4f/EMSatArcadia2.JPG" align="right"><br><br><br><br><br><br><br><br><br><br><br><br>

The cultures that were exposed to EMS for 45 minutes still showed colonies, meaning that the EMS was not strong enough to kill off the cells. Therefore, these colonies should theoretically show the most variation. The first fifty-five colonies that developed in both MG1655 and E-15 EG13 were compared at the 0 minute time point and the 45 minute time point using the Tecan (here's our <a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol#EMS">data</a>). <br>

2. Andrianantoandro, Ernesto, Subhayu Basu, David K. Karig, and Ron Weiss. "Synthetic biology: new engineering rules for an emerging discipline." Nature - Molecular Systems Biology. (2006): n. page. Web. 29 Aug. 2012. <http://www.nature.com/msb/journal/v2/n1/full/msb4100073.html>. <br>

3. Cupples, Claire, and Jeffrey Miller. "A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions." Proceedings of the National Academy of Sciences. 86. (1989): 5345-5349. Print.