http://2013.igem.org/wiki/index.php?title=Special:Contributions/Perry721&feed=atom&limit=50&target=Perry721&year=&month=2013.igem.org - User contributions [en]2024-03-29T00:37:44ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8_Characterization_of_Mutant_PhageTeam:BYU Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage2013-10-29T03:55:48Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
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{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: Experiments'''</font><br />
<br />
<br><br />
<br><br />
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|- valign="top"<br />
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<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
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| style="width: 82%; background-color: transparent;"|<br />
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<font face="Calibri" size="3"><br />
<br />
<font size="5"> '''10.8 Characterization of Mutant Phage''' </font><br />
<br />
<br><br />
<br />
'''I) Purpose'''<br />
<br />
: To characterize the mutant phage we selected from [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test]]<br />
<br />
'''II) Expected Outcome'''<br />
<br />
* EM pictures of phage with distinctively larger or smaller size.<br />
<br />
* Sequencing results that map out mutations that induce T7 phage capsid size changes.<br />
<br />
'''III) Reagents Used'''<br />
<br />
* Post-CsCl mutant phage from [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test]]<br />
<br />
* Mutant phage S4, S10, S21, and L8<br />
<br />
* x8 top agar<br />
<br />
* LB<br />
<br />
'''IV) Procedure'''<br />
<br />
1) Overview of previous attempts before iGEM Regional Jamboree<br />
<br />
* Before the Regional Jamboree, we identified our S4, S10, and L8 mutant. We tried to sequence their capsid genes (with the help of Dr. Grose) and take pictures using TEM. Unfortunately, the sequencing results' accuracy was less than 20%. And our phage does not have a high enough titer to be seen under electron microscope.<br />
<br />
* Thus we started off our characterization procedures with designing new primers (BI319 and BI320) and propagating our mutant phage.<br />
<br />
2) Propagating Mutant Phage (10.10)<br />
<br />
* Three different conditions of propagation was used for WT, S4, S10, and L8<br />
:: - Erlenmeyer flask: 10 mL LB + 1 mL E coli B liquid culture overnight + 10 uL of phage<br />
:: - 15 mL centrifuge tube: 5 mL LB + 0.5 mL E coli B liquid culture overnight + 10 uL of phage<br />
:: - autoclaved test tube: 1 mL of LB + 100 uL E coli B liquid culture overnight + 10 uL of phage<br />
<br />
* Wild-type and mutant phages were allowed to propagate for approximately 24 hours before purified via centrifugation and choloroform.<br />
<br />
* Spot tests at -2, -4, -6, and -8 was performed for each sample to estimated phage concentration after propagation.<br />
<br />
* Each phage sample was then plated at -7 using x4 agar to verify the mutants' phenotypic stability after propagation. <br />
<br />
3) TEM (10.16)<br />
<br />
* Phage Purification Team set up copper grids and arranged for TEM appointments to look at S4, S10 and L8<br />
<br />
4) Sequencing (10.17-10.?)<br />
<br />
* During the week when we were waiting for the new sequencing primers (BI319 and BI320) to arrive, we where able to isolate a new mutant phage S21. Thus, we will sequence S4, S10, S21, L8, and WT T7 phage to map out mutations altering phage capsid sizes.<br />
<br />
* DNA isolation, PCR, and gel electrophoresis protocol was similar to that of [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period2/Exp/5.20_T7_Minor_Capsid_Protein_PCR| 5.20 T7 Minor Capsid Protein PCR]]<br />
<br />
: ''Note for sequencing we used the primers BI257 and BI258.'' <br />
<br />
'''V) Results'''<br />
<br />
2) Propagating Mutant Phage<br />
<br />
* Plaque formed up to -6 for each sample, implying a concentration of at least 10E8 PFU/mL<br />
<br />
* Propagated phage retained their larger or smaller capsid size as confirmed by their smaller and larger plaque sizes at x4 agar.<br />
<br />
'''VI) Conclusion'''<br />
<br />
* We have taken several EM's of the phage, but have yet to get results for their capsid sizes. We hope to accomplish this before we leave for the jamboree.<br />
<br />
|}<br />
<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/File:Adv2.jpgFile:Adv2.jpg2013-10-29T03:52:54Z<p>Perry721: </p>
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<div></div>Perry721http://2013.igem.org/Team:BYU_Provo/TorontoTeam:BYU Provo/Toronto2013-10-29T03:52:42Z<p>Perry721: </p>
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<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''A Taste of Toronto'''</font><br />
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<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Toronto#Accomplishments|Accomplishments]]<br />
<br />
: [[Team:BYU_Provo/Toronto#Adventures|Adventures]]<br />
<br />
</font><br />
<br />
| style="width: 78%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Accomplishments==<br />
<br />
BYU 2013 iGEM Team is excited to announce that we won a gold medal during the North America Regional Jamboree and have advanced to the World Championships! We are especially proud because we are a team of undergraduate students, most of whom had limited research experience before iGEM, but we successfully competed in the over-graduate division. <br />
<br />
We really appreciate the marvelous work of iGEM headquarters and the University of Toronto for hosting us. We are also grateful for all the comments we received during our presentation and poster session. Your comments are invaluable to us and we will work hard to perfect Phage Pharming for the World Championships!<br />
<br />
==Adventures ==<br />
<br />
[[File:Adv1.jpg|550px|center|]]<br />
<br />
<br />
<br />
<br />
[[File:Adv2.jpg|550px|center|]]<br />
<br />
<br />
</font><br />
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{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/TorontoTeam:BYU Provo/Toronto2013-10-29T03:52:28Z<p>Perry721: </p>
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<div>{{TeamBYUProvo}}<br />
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<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''A Taste of Toronto'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Toronto#Accomplishments|Accomplishments]]<br />
<br />
: [[Team:BYU_Provo/Toronto#Adventures|Adventures]]<br />
<br />
</font><br />
<br />
| style="width: 78%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Accomplishments==<br />
<br />
BYU 2013 iGEM Team is excited to announce that we won a gold medal during the North America Regional Jamboree and have advanced to the World Championships! We are especially proud because we are a team of undergraduate students, most of whom had limited research experience before iGEM, but we successfully competed in the over-graduate division. <br />
<br />
We really appreciate the marvelous work of iGEM headquarters and the University of Toronto for hosting us. We are also grateful for all the comments we received during our presentation and poster session. Your comments are invaluable to us and we will work hard to perfect Phage Pharming for the World Championships!<br />
<br />
==Adventures ==<br />
<br />
[[File:Adv3.jpg|550px|center|]]<br />
<br />
<br />
<br />
<br />
[[File:Adv2.jpg|550px|center|]]<br />
<br />
<br />
</font><br />
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<br><br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/File:Adv1.jpgFile:Adv1.jpg2013-10-29T03:50:37Z<p>Perry721: </p>
<hr />
<div></div>Perry721http://2013.igem.org/Team:BYU_Provo/TorontoTeam:BYU Provo/Toronto2013-10-29T03:49:57Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''A Taste of Toronto'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Toronto#Accomplishments|Accomplishments]]<br />
<br />
: [[Team:BYU_Provo/Toronto#Adventures|Adventures]]<br />
<br />
</font><br />
<br />
| style="width: 78%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Accomplishments==<br />
<br />
BYU 2013 iGEM Team is excited to announce that we won a gold medal during the North America Regional Jamboree and have advanced to the World Championships! We are especially proud because we are a team of undergraduate students, most of whom had limited research experience before iGEM, but we successfully competed in the over-graduate division. <br />
<br />
We really appreciate the marvelous work of iGEM headquarters and the University of Toronto for hosting us. We are also grateful for all the comments we received during our presentation and poster session. Your comments are invaluable to us and we will work hard to perfect Phage Pharming for the World Championships!<br />
<br />
==Adventures ==<br />
<br />
[[File:Adv1.jpg|550px|center|]]<br />
<br />
<br />
<br />
<br />
[[File:Adv2.jpg|550px|center|]]<br />
<br />
<br />
</font><br />
<br />
<br><br />
<br><br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/TorontoTeam:BYU Provo/Toronto2013-10-29T03:49:44Z<p>Perry721: </p>
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{| width="100%"<br />
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<br />
: '''A Taste of Toronto'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Toronto#Accomplishments|Accomplishments]]<br />
<br />
: [[Team:BYU_Provo/Toronto#Adventures|Adventures]]<br />
<br />
</font><br />
<br />
| style="width: 78%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Accomplishments==<br />
<br />
BYU 2013 iGEM Team is excited to announce that we won a gold medal during the North America Regional Jamboree and have advanced to the World Championships! We are especially proud because we are a team of undergraduate students, most of whom had limited research experience before iGEM, but we successfully competed in the over-graduate division. <br />
<br />
We really appreciate the marvelous work of iGEM headquarters and the University of Toronto for hosting us. We are also grateful for all the comments we received during our presentation and poster session. Your comments are invaluable to us and we will work hard to perfect Phage Pharming for the World Championships!<br />
<br />
==Adventures ==<br />
<br />
[[File:Adv1.jpg|550px|center|]]<br />
<br />
[[File:Adv2.jpg|550px|center|]]<br />
<br />
<br />
</font><br />
<br />
<br><br />
<br><br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/TorontoTeam:BYU Provo/Toronto2013-10-29T03:49:30Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''A Taste of Toronto'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Toronto#Accomplishments|Accomplishments]]<br />
<br />
: [[Team:BYU_Provo/Toronto#Adventures|Adventures]]<br />
<br />
</font><br />
<br />
| style="width: 78%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Accomplishments==<br />
<br />
BYU 2013 iGEM Team is excited to announce that we won a gold medal during the North America Regional Jamboree and have advanced to the World Championships! We are especially proud because we are a team of undergraduate students, most of whom had limited research experience before iGEM, but we successfully competed in the over-graduate division. <br />
<br />
We really appreciate the marvelous work of iGEM headquarters and the University of Toronto for hosting us. We are also grateful for all the comments we received during our presentation and poster session. Your comments are invaluable to us and we will work hard to perfect Phage Pharming for the World Championships!<br />
<br />
==Adventures ==<br />
<br />
[[File:Adv1.jpg|550px|center|]]<br />
<br />
[[File:Adv2.jpg|550px|center|]]<br />
<br />
[[Team:BYU_Provo/Toronto/FixTest]]<br />
<br />
<br />
</font><br />
<br />
<br><br />
<br><br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/PartsTeam:BYU Provo/Parts2013-10-29T03:34:05Z<p>Perry721: </p>
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{{TeamBYUProvo}}<br />
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<br><br />
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{| width="100%"<br />
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: '''Parts Submitted to the Registry'''</font><br />
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|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
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<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Results|Results Overview]]<br />
<br />
: [[Team:BYU_Provo/Results/Judge|Judging Criteria]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Bronze|Bronze]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Silver|Silver]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Gold|Gold]]<br />
<br />
: [[Team:BYU_Provo/Results/Experimental|Experimental Results]]<br />
<br />
: [[Team:BYU_Provo/Results/Modeling|Modeling Results]]<br />
<br />
: [[Team:BYU_Provo/Parts|Parts Submitted]]<br />
<br />
</font><br />
<br />
| style="width: 81%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Parts Listing==<br />
<br />
An important aspect of the iGEM competition is the use and creation of standard biological parts. This page records the parts we plan to submit or have submitted to the registry this year. Details of each part and characterization data can be found on the respective parts.<br />
<br />
Parts submitted to the registry: BBa_K1195000, BBa_K1195001, BBa_K1195003,BBa_K1195004, BBa_K1195005, BBa_K1195009, BBa_K1195010, BBa_K1195011, and BBa_K1195012.<br />
<br />
Parts in preparation for submission to the registry: BBa_K1195002, BBa_K1195006<br />
<br />
Parts in the process of being fixed: BBa_K1195007, BBa_K1195008<br />
<br />
<groupparts>iGEM013 BYU_Provo</groupparts><br />
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{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/PartsTeam:BYU Provo/Parts2013-10-29T03:33:35Z<p>Perry721: </p>
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{{TeamBYUProvo}}<br />
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<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''Parts Submitted to the Registry'''</font><br />
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|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Results|Results Overview]]<br />
<br />
: [[Team:BYU_Provo/Results/Judge|Judging Criteria]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Bronze|Bronze]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Silver|Silver]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Gold|Gold]]<br />
<br />
: [[Team:BYU_Provo/Results/Experimental|Experimental Results]]<br />
<br />
: [[Team:BYU_Provo/Results/Modeling|Modeling Results]]<br />
<br />
: [[Team:BYU_Provo/Parts|Parts Submitted]]<br />
<br />
</font><br />
<br />
| style="width: 81%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Parts Listing==<br />
<br />
An important aspect of the iGEM competition is the use and creation of standard biological parts. This page records the parts we plan to submit or have submitted to the registry this year. Details of each part and characterization data can be found on the respective parts.<br />
<br />
Parts submitted to the registry: BBa_K1195000, BBa_K1195001, BBa_K1195003,BBa_K1195004, BBa_K1195005, BBa_K1195009, BBa_K1195010, BBa_K1195011, and BBa_K1195012.<br />
<br />
Parts in preparation for submission to the registry: BBa_1195002, BBa_1195006<br />
<br />
Parts in the process of being fixed: BBa_1195007, BBa_1195008<br />
<br />
<groupparts>iGEM013 BYU_Provo</groupparts><br />
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{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18_Cloning_T7_Capsid_Protein_into_iGEM_RegistryTeam:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry2013-10-29T03:31:51Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: Experiments'''</font><br />
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<br><br />
<br><br />
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|- valign="top"<br />
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<font color="#333399" size="3" font face="Calibri"><br />
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<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
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| style="width: 82%; background-color: transparent;"|<br />
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<font face="Calibri" size="3"><br />
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<font size="5"> '''10.18 Cloning T7 Capsid Protein into iGEM Registry''' </font><br />
<br />
<br><br />
<br />
'''I) Purpose'''<br />
<br />
: Clone both WT and mutant T7 Capsid Protein into iGEM registry<br />
<br />
'''II) Expected Outcome'''<br />
<br />
* WT and mutant T7 capsid protein cloned into the iGEM registry.<br />
<br />
'''III) Reagents Used'''<br />
<br />
* Reagents required for cloning: dNTP, Phusion polymerase, butter, ddH2O, etc.<br />
<br />
* Mutant phage S4, S10, S21, and L8; WT phage<br />
<br />
'''IV) Procedure'''<br />
<br />
1) Amplification and purification of insert (10.18)<br />
<br />
: I. DNA purification<br />
:: * Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling.<br />
:: * Boil for 12 minutes in the PCR machine (98 Celsius).<br />
:: * Remove the tubes from the PCR machine and keep on ice until use in PCR.<br />
<br />
: II. PCR w/ Phusion polymerase<br />
:: * To a eppendorf tube, add (for 6 PCR reactions simultaneously):<br />
::: - 210 uL of ddH2O<br />
::: - 60uL 5x Phusion buffer<br />
::: - 9uL 10mM dNTPs<br />
::: - 6uL of each primer (BI309 and BI310) <br />
:: * Mix well<br />
:: * To six new PCR tubes, labeled S4, S10, S21, L8, WT, and control respectively, add 2uL of template DNA from the boiled samples. <br />
:: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix).<br />
:: * Add 48uL of the master mix into each PCR tube.<br />
:: * Run 35 cycles with temperatures of 95 C, 60 C, and 72 C with an extension time of 2 minutes.<br />
:: * Leave overnight in the freezer.<br />
<br />
: III. Low melt gel electrophoresis (10.19)<br />
:: * Add 75 ml of 1X TAE buffer and 0.75 grams of low melt agarose to an Erlenmeyer flask. Put into the microwave for about 90 seconds or until the agarose is completely dissolved. 1 drop of ethidium bromide was then added and mixed with the gel.<br />
:: * Pour the liquid onto the gel bed and let it cool. Remember to insert a sample comb.<br />
:: * The gel allowed to sit for overnight in the fridge to solidify.<br />
:: * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts.<br />
<br />
2) Repeat of the amplification and purification of insert (10.20)<br />
: Protocols of this experiment were similar to that of 1) Amplification and purification of insert (10.18), except for the following differences<br />
:: * 10uL of phage and 40uL of ddH2O was used in the boiling process<br />
:: * extention time was changed to 3 miinutes<br />
:: * a regular gel was used to verify PCR product<br />
:: * 3uL of product and 3uL of loading dye was used per well in the gel.<br />
<br />
3) Restriction digest (10.21)<br />
: * Followed procedure for PCR cleanup of WT, S4, S10, S21, and L8.<br />
: * For each PCR product (WT, S4, S10, S21, and L8), we set up an individual tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of PCR product, and 2ul of each restriction enzyme.<br />
: * We also made a vector digestion tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of the vector digestion, and 2ul of each restriction enzyme.<br />
: * The six tubes were placed in the 37C incubator overnight.<br />
<br />
4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22)<br />
: * Ran a low-melt gel for each of the tubes in step 3.<br />
: * Cut out each bright band and placed them in an eppendorf tube.<br />
: * Left them in the freezer overnight.<br />
<br />
5) Ligation (10.23)<br />
: * Centrifuged the eppendorf tubes from step 4 to separate the vector or insert from the gel.<br />
: * Created 5 ligation reactions for each phage (WT, S4, S10, S21, and L8) with 6.5ul water, 1.5ul 10X ligase buffer, 1ul T4 DNA ligase, 3ul vector, and 3ul insert.<br />
<br />
6) Transformation (10.23)<br />
: * Added 2ul of ligation mix to 25ul of competent cells. Vortexed the tubes briefly and placed them on ice for 10 minutes.<br />
: * Heat shocked at 42C for 1 minute and then placed tubes back on ice for 10 minutes.<br />
: * Added 0.5mL of plain LB to the reactions and incubated at 37C for 1 hour.<br />
: * Plated 100ul of cells on CAM plates and incubated at 37C overnight.<br />
<br />
7) Colony PCR and Overnights for Vector Purification (10.24)<br />
: * Our transformations only had WTa, WTb, S4, S10a, S10b, S10c, S21, L8a, and L8b (a,b,c designates multiple colonies from the same transformation.<br />
: * For each colony, we picked it with a pipet tip, dipped it in 50ul of water in a PCR tube, and placed the tip in a test tube of 5mL of LB (These tubes were placed in the 37C incubator as overnights).<br />
: * Boil each PCR tube for 5 minutes.<br />
: * Set up a PCR reaction for each PCR tube by adding 2.5ul Standard 10X reaction buffer, 0.5ul 10mM dNTP's, 0.5ul of each primer, 0.5ul Taq DNA polymerase, and 2ul of boiled colony sample.<br />
: * Run PCR for 25 cycles.<br />
: * Run a gel with 5ul of each PCR product to check for proper size.<br />
<br />
8) Plasmid Cleanup (10.25)<br />
: * Followed procedure for plasmid cleanup for WT, S4, S10, and L8.<br />
: * Mailed 10ul of each plasmid to iGEM!<br />
<br />
Monday: Restriction digest<br />
Tuesday: Low-melt gel for purification of sticky-ended insert/vector (Jade)<br />
Wednesday: Ligation and transformation<br />
Thursday: Redid plating for transformation and started overnights of each colony and ran gel<br />
Friday: Cleaned up the plasmid and sent it in<br />
<br />
'''V) Results'''<br />
<br />
1) Amplification and purification of insert <br />
: Only WT and S10 showed bands at approximately 1.1k bp. This experiment needs to be repeated <br />
<br />
<br />
'''VI) Conclusion'''<br />
<br />
: The cloning worked for WT, S4, S10, and L8. We submitted all these parts to the registry.<br />
|}<br />
<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/File:Asdfsa1.JPGFile:Asdfsa1.JPG2013-10-29T03:28:18Z<p>Perry721: </p>
<hr />
<div></div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/FallexpTeam:BYU Provo/Notebook/SmallPhage/Fallexp2013-10-29T03:27:29Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 50%; background-color: transparent;"|<br />
<br />
<font size="5" font face="Calibri"> '''September 1 - September 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> While the phage purification team was working out a kink in their cesium chloride gradient, we focused on modeling the relationship between capsid size and plaque size. Then once the cesium chloride gradient was perfected, we performed another round of mutagenesis, hoping that we would now be able to see mutant T7 bacteriophage. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''September 16 - September 30''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> All our hard work finally paid off! We managed to isolate a smaller and a larger T7 bacteriophage! </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period2/Dailylog|Daily log]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:BYUSPFall2.JPG|220px|center|link=]]<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''October 1 - October 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> We had a great time at the iGEM regionals in Toronto! We're excited to be going to the World Championship Jamboree and got right back to work. Our first focus was getting good pictures of our mutant phage so that we could confirm their phenotypes. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa1.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''October 16 - October 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> For the last weeks of iGEM, we continued to characterize the mutant phage more. We sequenced the capsid protein and searched for mutations. In addition, we cloned in the capsid proteins for T7 wild type, S4 (Small mutant 1), S10 (Small mutant 2), and L8 (Large Mutant 1). </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa2.JPG|220px|center]]<br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period4/Dailylog2013-10-29T03:25:20Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: October 16 - October 31 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''10/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Plated for mutants as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed amplification and purification of insert as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/19/13''' </font><br />
<br />
JL, LP<br />
<br />
* Ran a low melt gel for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/20/13''' </font><br />
<br />
JL<br />
<br />
* Performed the repeat PCR and gel step in [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/21/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did restriction digest for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/22/13''' </font><br />
<br />
JL<br />
<br />
* Low-melt gel for purification of sticky-ended insert/vector for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/23/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did ligation and transformation for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/24/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did Colony PCR and Overnights for Vector Purification for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/25/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did plasmid cleanup for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]] and sent the plasmid into iGEM!<br />
<br />
<br><br />
<br />
<font size="4"> '''10/27/13''' </font><br />
<br />
JL<br />
<br />
* Started PCR reaction for sequencing WT, S4, S10, S21, S22, and L8 for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/28/13''' </font><br />
<br />
JL, LP<br />
<br />
* Propagated WT, S4, S10, S21, S22, and L8 for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]]<br />
<br />
* Started PCR reaction for sequencing WT, S4, S10, S21, S22, and L8 for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Mapped out mutation for S4 capsid protein.<br />
<br />
* Finished the wiki.<br />
<br />
<br><br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period4/Dailylog2013-10-29T03:24:59Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: October 16 - October 31 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''10/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Plated for mutants as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed amplification and purification of insert as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/19/13''' </font><br />
<br />
JL, LP<br />
<br />
* Ran a low melt gel for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/20/13''' </font><br />
<br />
JL<br />
<br />
* Performed the repeat PCR and gel step in [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/21/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did restriction digest for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/22/13''' </font><br />
<br />
JL<br />
<br />
* Low-melt gel for purification of sticky-ended insert/vector for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/23/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did ligation and transformation for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/24/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did Colony PCR and Overnights for Vector Purification for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/25/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did plasmid cleanup for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]] and sent the plasmid into iGEM!<br />
<br />
<br><br />
<br />
<font size="4"> '''10/27/13''' </font><br />
<br />
JL<br />
<br />
* Started PCR reaction for sequencing WT, S4, S10, S21, S22, and L8 for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/28/13''' </font><br />
<br />
JL<br />
<br />
* Propagated WT, S4, S10, S21, S22, and L8 for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]]<br />
<br />
JL, LP<br />
<br />
* Started PCR reaction for sequencing WT, S4, S10, S21, S22, and L8 for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Mapped out mutation for S4 capsid protein.<br />
<br />
* Finished the wiki.<br />
<br />
<br><br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period4/Dailylog2013-10-29T03:24:27Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: October 16 - October 31 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''10/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Plated for mutants as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed amplification and purification of insert as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/19/13''' </font><br />
<br />
JL, LP<br />
<br />
* Ran a low melt gel for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/20/13''' </font><br />
<br />
JL<br />
<br />
* Performed the repeat PCR and gel step in [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/21/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did restriction digest for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/22/13''' </font><br />
<br />
JL<br />
<br />
* Low-melt gel for purification of sticky-ended insert/vector for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/23/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did ligation and transformation for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/24/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did Colony PCR and Overnights for Vector Purification for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]]<br />
<br />
<br><br />
<br />
<font size="4"> '''10/25/13''' </font><br />
<br />
JL, LP<br />
<br />
* Did plasmid cleanup for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|10.18 Cloning T7 Capsid Protein into iGEM Registry]] and sent the plasmid into iGEM!<br />
<br />
<br><br />
<br />
<font size="4"> '''10/27/13''' </font><br />
<br />
JL<br />
<br />
* Started PCR reaction for sequencing WT, S4, S10, S21, S22, and L8 for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/28/13''' </font><br />
<br />
JL<br />
<br />
* Propagated WT, S4, S10, S21, S22, and L8 for <font size="4"> '''10/27/13''' </font><br />
<br />
JL, LP<br />
<br />
* Started PCR reaction for sequencing WT, S4, S10, S21, S22, and L8 for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Mapped out mutation for S4 capsid protein.<br />
<br />
* Finished the wiki.<br />
<br />
<br><br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18_Cloning_T7_Capsid_Protein_into_iGEM_RegistryTeam:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry2013-10-29T03:15:14Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: Experiments'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="5"> '''10.18 Cloning T7 Capsid Protein into iGEM Registry''' </font><br />
<br />
<br><br />
<br />
'''I) Purpose'''<br />
<br />
: Clone both WT and mutant T7 Capsid Protein into iGEM registry<br />
<br />
'''II) Expected Outcome'''<br />
<br />
* WT and mutant T7 capsid protein cloned into the iGEM registry.<br />
<br />
'''III) Reagents Used'''<br />
<br />
* Reagents required for cloning: dNTP, Phusion polymerase, butter, ddH2O, etc.<br />
<br />
* Mutant phage S4, S10, S21, and L8; WT phage<br />
<br />
'''IV) Procedure'''<br />
<br />
1) Amplification and purification of insert (10.18)<br />
<br />
: I. DNA purification<br />
:: * Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling.<br />
:: * Boil for 12 minutes in the PCR machine (98 Celsius).<br />
:: * Remove the tubes from the PCR machine and keep on ice until use in PCR.<br />
<br />
: II. PCR w/ Phusion polymerase<br />
:: * To a eppendorf tube, add (for 6 PCR reactions simultaneously):<br />
::: - 210 uL of ddH2O<br />
::: - 60uL 5x Phusion buffer<br />
::: - 9uL 10mM dNTPs<br />
::: - 6uL of each primer (BI309 and BI310) <br />
:: * Mix well<br />
:: * To six new PCR tubes, labeled S4, S10, S21, L8, WT, and control respectively, add 2uL of template DNA from the boiled samples. <br />
:: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix).<br />
:: * Add 48uL of the master mix into each PCR tube.<br />
:: * Run 35 cycles with temperatures of 95 C, 60 C, and 72 C with an extension time of 2 minutes.<br />
:: * Leave overnight in the freezer.<br />
<br />
: III. Low melt gel electrophoresis (10.19)<br />
:: * Add 75 ml of 1X TAE buffer and 0.75 grams of low melt agarose to an Erlenmeyer flask. Put into the microwave for about 90 seconds or until the agarose is completely dissolved. 1 drop of ethidium bromide was then added and mixed with the gel.<br />
:: * Pour the liquid onto the gel bed and let it cool. Remember to insert a sample comb.<br />
:: * The gel allowed to sit for overnight in the fridge to solidify.<br />
:: * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts.<br />
<br />
2) Repeat of the amplification and purification of insert (10.20)<br />
: Protocols of this experiment were similar to that of 1) Amplification and purification of insert (10.18), except for the following differences<br />
:: * 10uL of phage and 40uL of ddH2O was used in the boiling process<br />
:: * extention time was changed to 3 miinutes<br />
:: * a regular gel was used to verify PCR product<br />
:: * 3uL of product and 3uL of loading dye was used per well in the gel.<br />
<br />
3) Restriction digest (10.21)<br />
: * Followed procedure for PCR cleanup of WT, S4, S10, S21, and L8.<br />
: * For each PCR product (WT, S4, S10, S21, and L8), we set up an individual tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of PCR product, and 2ul of each restriction enzyme.<br />
: * We also made a vector digestion tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of the vector digestion, and 2ul of each restriction enzyme.<br />
: * The six tubes were placed in the 37C incubator overnight.<br />
<br />
4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22)<br />
: * Ran a low-melt gel for each of the tubes in step 3.<br />
: * Cut out each bright band and placed them in an eppendorf tube.<br />
: * Left them in the freezer overnight.<br />
<br />
5) Ligation (10.23)<br />
: * Centrifuged the eppendorf tubes from step 4 to separate the vector or insert from the gel.<br />
: * Created 5 ligation reactions for each phage (WT, S4, S10, S21, and L8) with 6.5ul water, 1.5ul 10X ligase buffer, 1ul T4 DNA ligase, 3ul vector, and 3ul insert.<br />
<br />
6) Transformation (10.23)<br />
: * Added 2ul of ligation mix to 25ul of competent cells. Vortexed the tubes briefly and placed them on ice for 10 minutes.<br />
: * Heat shocked at 42C for 1 minute and then placed tubes back on ice for 10 minutes.<br />
: * Added 0.5mL of plain LB to the reactions and incubated at 37C for 1 hour.<br />
: * Plated 100ul of cells on CAM plates and incubated at 37C overnight.<br />
<br />
7) Colony PCR and Overnights for Vector Purification (10.24)<br />
: * Our transformations only had WTa, WTb, S4, S10a, S10b, S10c, S21, L8a, and L8b (a,b,c designates multiple colonies from the same transformation.<br />
: * For each colony, we picked it with a pipet tip, dipped it in 50ul of water in a PCR tube, and placed the tip in a test tube of 5mL of LB (These tubes were placed in the 37C incubator as overnights).<br />
: * Boil each PCR tube for 5 minutes.<br />
: * Set up a PCR reaction for each PCR tube by adding 2.5ul Standard 10X reaction buffer, 0.5ul 10mM dNTP's, 0.5ul of each primer, 0.5ul Taq DNA polymerase, and 2ul of boiled colony sample.<br />
: * Run PCR for 25 cycles.<br />
: * Run a gel with 5ul of each PCR product to check for proper size.<br />
<br />
8) Plasmid Cleanup (10.25)<br />
: * Followed procedure for plasmid cleanup for WT, S4, S10, and L8.<br />
: * Mailed 10ul of each plasmid to iGEM!<br />
<br />
Monday: Restriction digest<br />
Tuesday: Low-melt gel for purification of sticky-ended insert/vector (Jade)<br />
Wednesday: Ligation and transformation<br />
Thursday: Redid plating for transformation and started overnights of each colony and ran gel<br />
Friday: Cleaned up the plasmid and sent it in<br />
<br />
'''V) Results'''<br />
<br />
1) Amplification and purification of insert <br />
: Only WT and S10 showed bands at approximately 1.1k bp. This experiment needs to be repeated <br />
<br />
<br />
'''VI) Conclusion'''<br />
<br />
|}<br />
<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18_Cloning_T7_Capsid_Protein_into_iGEM_RegistryTeam:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry2013-10-29T02:46:19Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: Experiments'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="5"> '''10.18 Cloning T7 Capsid Protein into iGEM Registry''' </font><br />
<br />
<br><br />
<br />
'''I) Purpose'''<br />
<br />
: Clone both WT and mutant T7 Capsid Protein into iGEM registry<br />
<br />
'''II) Expected Outcome'''<br />
<br />
* WT and mutant T7 capsid protein cloned into the iGEM registry.<br />
<br />
'''III) Reagents Used'''<br />
<br />
* Reagents required for cloning: dNTP, Phusion polymerase, butter, ddH2O, etc.<br />
<br />
* Mutant phage S4, S10, S21, and L8; WT phage<br />
<br />
'''IV) Procedure'''<br />
<br />
1) Amplification and purification of insert (10.18)<br />
<br />
: I. DNA purification<br />
:: * Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling.<br />
:: * Boil for 12 minutes in the PCR machine (98 Celsius).<br />
:: * Remove the tubes from the PCR machine and keep on ice until use in PCR.<br />
<br />
: II. PCR w/ Phusion polymerase<br />
:: * To a eppendorf tube, add (for 6 PCR reactions simultaneously):<br />
::: - 210 uL of ddH2O<br />
::: - 60uL 5x Phusion buffer<br />
::: - 9uL 10mM dNTPs<br />
::: - 6uL of each primer (BI309 and BI310) <br />
:: * Mix well<br />
:: * To six new PCR tubes, labeled S4, S10, S21, L8, WT, and control respectively, add 2uL of template DNA from the boiled samples. <br />
:: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix).<br />
:: * Add 48uL of the master mix into each PCR tube.<br />
:: * Run 35 cycles with temperatures of 95 C, 60 C, and 72 C with an extension time of 2 minutes.<br />
:: * Leave overnight in the freezer.<br />
<br />
: III. Low melt gel electrophoresis (10.19)<br />
:: * Add 75 ml of 1X TAE buffer and 0.75 grams of low melt agarose to an Erlenmeyer flask. Put into the microwave for about 90 seconds or until the agarose is completely dissolved. 1 drop of ethidium bromide was then added and mixed with the gel.<br />
:: * Pour the liquid onto the gel bed and let it cool. Remember to insert a sample comb.<br />
:: * The gel allowed to sit for overnight in the fridge to solidify.<br />
:: * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts.<br />
<br />
2) Repeat of the amplification and purification of insert (10.20)<br />
: Protocols of this experiment were similar to that of 1) Amplification and purification of insert (10.18), except for the following differences<br />
:: * 10uL of phage and 40uL of ddH2O was used in the boiling process<br />
:: * extention time was changed to 3 miinutes<br />
:: * a regular gel was used to verify PCR product<br />
:: * 3uL of product and 3uL of loading dye was used per well in the gel.<br />
<br />
3) Restriction digest (10.21)<br />
: * Followed procedure for PCR cleanup of WT, S4, S10, S21, and L8.<br />
: * For each PCR product (WT, S4, S10, S21, and L8), we set up an individual tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of PCR product, and 2ul of each restriction enzyme.<br />
: * We also made a vector digestion tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of the vector digestion, and 2ul of each restriction enzyme.<br />
: * The six tubes were placed in the 37C incubator overnight.<br />
<br />
4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22)<br />
: * Ran a low-melt gel for each of the tubes in step 3.<br />
: * Cut out each bright band and placed them in an eppendorf tube.<br />
: * Left them in the freezer overnight.<br />
<br />
5) Ligation (10.23)<br />
: * Centrifuged the eppendorf tubes from step 4 to separate the vector or insert from the gel.<br />
: * Created 5 ligation reactions for each phage (WT, S4, S10, S21, and L8) with 6.5ul water, 1.5ul 10X ligase buffer, 1ul T4 DNA ligase, 3ul vector, and 3ul insert.<br />
<br />
6) Transformation (<br />
<br />
Monday: Restriction digest<br />
Tuesday: Low-melt gel for purification of sticky-ended insert/vector (Jade)<br />
Wednesday: Ligation and transformation<br />
Thursday: Redid plating for transformation and started overnights of each colony and ran gel<br />
Friday: Cleaned up the plasmid and sent it in<br />
<br />
'''V) Results'''<br />
<br />
1) Amplification and purification of insert <br />
: Only WT and S10 showed bands at approximately 1.1k bp. This experiment needs to be repeated <br />
<br />
<br />
'''VI) Conclusion'''<br />
<br />
|}<br />
<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18_Cloning_T7_Capsid_Protein_into_iGEM_RegistryTeam:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry2013-10-29T02:42:46Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: Experiments'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="5"> '''10.18 Cloning T7 Capsid Protein into iGEM Registry''' </font><br />
<br />
<br><br />
<br />
'''I) Purpose'''<br />
<br />
: Clone both WT and mutant T7 Capsid Protein into iGEM registry<br />
<br />
'''II) Expected Outcome'''<br />
<br />
* WT and mutant T7 capsid protein cloned into the iGEM registry.<br />
<br />
'''III) Reagents Used'''<br />
<br />
* Reagents required for cloning: dNTP, Phusion polymerase, butter, ddH2O, etc.<br />
<br />
* Mutant phage S4, S10, S21, and L8; WT phage<br />
<br />
'''IV) Procedure'''<br />
<br />
1) Amplification and purification of insert (10.18)<br />
<br />
: I. DNA purification<br />
:: * Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling.<br />
:: * Boil for 12 minutes in the PCR machine (98 Celsius).<br />
:: * Remove the tubes from the PCR machine and keep on ice until use in PCR.<br />
<br />
: II. PCR w/ Phusion polymerase<br />
:: * To a eppendorf tube, add (for 6 PCR reactions simultaneously):<br />
::: - 210 uL of ddH2O<br />
::: - 60uL 5x Phusion buffer<br />
::: - 9uL 10mM dNTPs<br />
::: - 6uL of each primer (BI309 and BI310) <br />
:: * Mix well<br />
:: * To six new PCR tubes, labeled S4, S10, S21, L8, WT, and control respectively, add 2uL of template DNA from the boiled samples. <br />
:: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix).<br />
:: * Add 48uL of the master mix into each PCR tube.<br />
:: * Run 35 cycles with temperatures of 95 C, 60 C, and 72 C with an extension time of 2 minutes.<br />
:: * Leave overnight in the freezer.<br />
<br />
: III. Low melt gel electrophoresis (10.19)<br />
:: * Add 75 ml of 1X TAE buffer and 0.75 grams of low melt agarose to an Erlenmeyer flask. Put into the microwave for about 90 seconds or until the agarose is completely dissolved. 1 drop of ethidium bromide was then added and mixed with the gel.<br />
:: * Pour the liquid onto the gel bed and let it cool. Remember to insert a sample comb.<br />
:: * The gel allowed to sit for overnight in the fridge to solidify.<br />
:: * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts.<br />
<br />
2) Repeat of the amplification and purification of insert (10.20)<br />
: Protocols of this experiment were similar to that of 1) Amplification and purification of insert (10.18), except for the following differences<br />
:: * 10uL of phage and 40uL of ddH2O was used in the boiling process<br />
:: * extention time was changed to 3 miinutes<br />
:: * a regular gel was used to verify PCR product<br />
:: * 3uL of product and 3uL of loading dye was used per well in the gel.<br />
<br />
3) Restriction digest (10.21)<br />
: * Followed procedure for PCR cleanup of WT, S4, S10, S21, and L8.<br />
: * For each PCR product (WT, S4, S10, S21, and L8), we set up an individual tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of PCR product, and 2ul of each restriction enzyme.<br />
: * We also made a vector digestion tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of the vector digestion, and 2ul of each restriction enzyme.<br />
: * We added part of the vector digestion tube to each of the individual tubes.<br />
: * The five individual tubes were placed in the 37C incubator overnight.<br />
<br />
4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22)<br />
: * Ran a low-melt gel for each of the individual tubes.<br />
: * Cut out each bright band and placed them in an eppendorf tube.<br />
: * Left them in the freezer overnight.<br />
<br />
5) Ligation (10.23)<br />
: * Centrifuged the eppendorf tubes from step 4 to separate the <br />
<br />
Monday: Restriction digest<br />
Tuesday: Low-melt gel for purification of sticky-ended insert/vector (Jade)<br />
Wednesday: Ligation and transformation<br />
Thursday: Redid plating for transformation and started overnights of each colony and ran gel<br />
Friday: Cleaned up the plasmid and sent it in<br />
<br />
'''V) Results'''<br />
<br />
1) Amplification and purification of insert <br />
: Only WT and S10 showed bands at approximately 1.1k bp. This experiment needs to be repeated <br />
<br />
<br />
'''VI) Conclusion'''<br />
<br />
|}<br />
<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/FallexpTeam:BYU Provo/Notebook/SmallPhage/Fallexp2013-10-29T01:55:36Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 50%; background-color: transparent;"|<br />
<br />
<font size="5" font face="Calibri"> '''September 1 - September 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> While the phage purification team was working out a kink in their cesium chloride gradient, we focused on modeling the relationship between capsid size and plaque size. Then once the cesium chloride gradient was perfected, we performed another round of mutagenesis, hoping that we would now be able to see mutant T7 bacteriophage. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''September 16 - September 30''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> All our hard work finally paid off! We managed to isolate a smaller and a larger T7 bacteriophage! </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period2/Dailylog|Daily log]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:BYUSPFall2.JPG|220px|center|link=]]<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''October 1 - October 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> We had a great time at the iGEM regionals in Toronto! We're excited to be going to the World Championship Jamboree and got right back to work. Our first focus was getting good pictures of our mutant phage so that we could confirm their phenotypes. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''October 16 - October 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> For the last weeks of iGEM, we continued to characterize the mutant phage more. We sequenced the capsid protein and searched for mutations. In addition, we cloned in the capsid proteins for T7 wild type, S4 (Small mutant 1), S10 (Small mutant 2), and L8 (Large Mutant 1). </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/ResultsTeam:BYU Provo/Results2013-10-28T23:58:59Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
[[File:BYUResults.JPG|965px|center|link=Team:BYU_Provo/Results/Experimental]]<br />
<br />
{| width="100%" style="text-align: top;"<br />
<br />
|- valign="top"<br />
| style="width: 56%; background-color: transparent;"|<br />
<br />
<br><br />
<br />
<font color="#333399" size="5" font face="Calibri"> <center> Achievements </center></font><br />
<br />
<br><br />
<br />
<font color="#333399" size="3" font face="Calibri"> <br />
<br />
'''Phage Library'''<br />
<br />
This summer, we developed a cesium chloride gradient protocol for isolating larger and smaller phage. Utilizing this gradient, we were able to isolate mutant T4 bacteriophages that have distinctively smaller capsids and less variability in capsid size when compared to the wild type. Similarly, we were also able to isolate mutant T7 bacteriophages that have smaller and larger than average capsid sizes.<br />
<br />
<br><br />
<br />
'''Cholera'''<br />
<br />
To tackle the disease Cholera, we identified a protein, Amylase, that disrupt Cholera's biofilm formation. In addition, we demonstrated that ''E. coli'' is capable of sensing Cholera and hypothesize that it does so through its SdiA protein. In fact, Cholera can induce Lambda from lysogenic ''E. coli''.<br />
<br />
</font><br />
<br />
| style="width: 22%; background-color: transparent;"|<br />
<br><br />
<br><br />
<font color="#333399" size="5" font face="Calibri"> <center>[[Team:BYU_Provo/Results/Judge|Judging Criteria]] </center></font><br />
<br><br />
<br><br />
<br />
[[File:BYUJudgeIcon.JPG|200px|center|link=Team:BYU_Provo/Results/Judge]]<br />
<br />
| style="width: 22%; background-color: transparent;"|<br />
<br><br />
<br><br />
<font color="#333399" size="5" font face="Calibri"> <center> Specifics </center></font><br />
<br><br />
<br><br />
<br />
{|<br />
<br />
|- valign="top"<br />
<br />
| style="background-color: transparent;"| [[File:BYUTeamExperimentalIconIcon.JPG|200px|center|link=Team:BYU_Provo/Results/Experimental]]<br />
<br />
|- <br />
<br />
| style="background-color: transparent;"| [[File:BYUTeamModelingIcon.JPG|200px|center|link=Team:BYU Provo/Results/Modeling]]<br />
<br />
|- <br />
<br />
| style="background-color: transparent;"| [[File:BYUTeamPartsIcon.JPG|200px|center|link=Team:BYU_Provo/Parts]]<br />
<br />
|}<br />
<br />
|}<br />
<br />
<br><br />
<br />
<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period3/Dailylog2013-10-20T23:54:34Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: October 1 - October 15 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''10/1/13 - 10/3/13''' </font><br />
<br />
JL, LP<br />
<br />
* Worked on putting together Jamboree presentation and poster. <br />
<br />
* Tried to take EM to verify mutant size - did not work very well: phage titer is too low<br />
<br />
<br><br />
<br />
<font size="4"> '''10/4/13 - 10/6/13''' </font><br />
<br />
JL, LP<br />
<br />
* North America Regional Jamboree! For more information click [[Team:BYU_Provo/Toronto|here]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/7/13''' </font><br />
<br />
JL<br />
<br />
* Started approximately 10 mL E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''10/8/13''' </font><br />
<br />
JL<br />
<br />
* Started [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]] by propagating the mutants.<br />
<br />
<br><br />
<br />
<font size="4"> '''10/9/13''' </font><br />
<br />
JL, LP<br />
<br />
* Purified the propagated mutant from [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''10/10/13''' </font><br />
<br />
LP<br />
<br />
* Performed spot tests to estimate the mutant phage titer in [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
JL<br />
<br />
* Designed primers for cloning the major and minor capsid proteins into the iGEM registry: BI309 (Xbal) and BI310 (SpeI)<br />
<br />
<br><br />
<br />
<font size="4"> '''10/11/13''' </font><br />
<br />
JL, LP<br />
<br />
* Plated the mutant phage at -7 to verify their phenotypic stability. For specifics, please see [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Designed primers for more accurate sequencing: BI319 (160bp upstream from start codon) and BI320 (-160 downstream from minor capsid protein stop codon)<br />
<br />
<br><br />
<br />
<font size="4"> '''10/12/13''' </font><br />
<br />
JL<br />
<br />
* Started approximately 15mL of E coli B liquid culture overnight<br />
<br />
<br><br />
<br />
<font size="4"> '''10/13/13''' </font><br />
<br />
JL<br />
<br />
* Plated more phage from CsCl gradient to select for mutant. Specifics are recorded in [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/14/13''' </font><br />
<br />
JL, LP<br />
<br />
* Plated more phage to look for mutants as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period4/Dailylog2013-10-20T23:52:43Z<p>Perry721: Created page with "{{TeamBYUProvo}} <br> {| width="100%" | colspan="3" | <font color="#333399" size="5" font face="Calibri"> : '''Small Phage September - October Notebook: October 16 - October ..."</p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: October 16 - October 31 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''10/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Plated for mutants as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed amplification and purification of insert as part of [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|Cloning T7 Capsid Protein into iGEM Registry]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/19/13''' </font><br />
<br />
JL, LP<br />
<br />
* Ran a low melt gel for [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry|Cloning T7 Capsid Protein into iGEM Registry]].<br />
<br />
<br><br />
<br />
<font size="4"> '''10/8/13''' </font><br />
<br />
JL<br />
<br />
* Started [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]] by propagating the mutants.<br />
<br />
<br><br />
<br />
<font size="4"> '''10/9/13''' </font><br />
<br />
JL, LP<br />
<br />
* Purified the propagated mutant from [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''10/10/13''' </font><br />
<br />
LP<br />
<br />
* Performed spot tests to estimate the mutant phage titer in [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
JL<br />
<br />
* Designed primers for cloning the major and minor capsid proteins into the iGEM registry: BI309 (Xbal) and BI310 (SpeI)<br />
<br />
<br><br />
<br />
<font size="4"> '''10/11/13''' </font><br />
<br />
JL, LP<br />
<br />
* Plated the mutant phage at -7 to verify their phenotypic stability. For specifics, please see [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
* Designed primers for more accurate sequencing: BI319 (160bp upstream from start codon) and BI320 (-160 downstream from minor capsid protein stop codon)<br />
<br />
<br><br />
<br />
<font size="4"> '''10/12/13''' </font><br />
<br />
JL<br />
<br />
* Started approximately 15mL of E coli B liquid culture overnight<br />
<br />
<br><br />
<br />
<font size="4"> '''10/13/13''' </font><br />
<br />
JL<br />
<br />
* Plated more phage from CsCl gradient to select for mutant. Specifics are recorded in [[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/10.8 Characterization of Mutant Phage|10.8 Characterization of Mutant Phage]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/27/13''' </font><br />
<br />
JL, LP<br />
<br />
* Determined results for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Finished updating the wiki.<br />
<br />
* Started approximately 30mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period3/Dailylog2013-10-09T21:37:57Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: October 1 - October 15 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''9/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed plans for these future two weeks: established priority.<br />
<br />
* Help Large Phage Group with dilution series and performing a spot test for their small phage band. <br />
<br />
<br><br />
<br />
<font size="4"> '''9/17/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed CsCl gradient set up with Phage Purification Team.<br />
<br />
* Performed dilution series and spot test for samples post CsCl from [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/19/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/20/13''' </font><br />
<br />
LP<br />
<br />
* Performed "Plating to Check for Plaque Size" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/23/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Plating to Check for Plaque Size 2" and "Checking for Plaque Viability from Step 5" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/24/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 30 mL E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/25/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Checking for Plaque Viability 2" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Started approximately 25mL of E coli B liquid culture overnight<br />
<br />
<br><br />
<br />
<font size="4"> '''9/26/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 25mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/27/13''' </font><br />
<br />
JL, LP<br />
<br />
* Determined results for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Finished updating the wiki.<br />
<br />
* Started approximately 30mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period1/Dailylog2013-10-09T21:37:38Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: September 1 - September 15 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''9/1/13''' </font><br />
<br />
* Performed additional spot tests for 12 samples to verify results of phage concentration in each aliquot for [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.26 Mutagen Concentration Test|8.26 Mutagen Concentration Test - Eighth Protocol]].<br />
<br />
* Started approximately 10mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/2/13''' </font><br />
<br />
* Worked on compiling data for [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.26 Mutagen Concentration Test|8.26 Mutagen Concentration Test - Eighth Protocol]]; there are significant differences between CsCl gradient for control sample and that for mutated phage sample.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/3/13''' </font><br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/4/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed results from recent spot test for [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.26 Mutagen Concentration Test|8.26 Mutagen Concentration Test - Eighth Protocol]]. Phage too spread out to effectively select for mutant phage. Decided to stop until the Phage Purification Team can get the wildtype T7 phage to band.<br />
<br />
* Discussed plans and designs for team wiki with Darren and Keltzie.<br />
<br />
* Started T7 propagation to generate enough phage for the Phage Purification Team to experiment with. Specifically, to a 15mL centrifuge tube we added 8mL of LB, 2mL of E coli B liquid culture overnight, and 20uL of 8.24 T7 phage stock.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/5/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 10mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/6/13''' </font><br />
<br />
JL, LP<br />
<br />
* Purified phage propagation sample from 9.4 via centrifugation (5 minutes at 3000 rpm) and cholorform. Spot tests were then performed to estimate phage titer.<br />
<br />
* Took pictures of plates from [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size|8.16 Modeling phage plaque size - Experiment One]] using alpha imager.<br />
<br />
* Divided up assignments for updating notebook.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/7/13''' </font><br />
<br />
JL<br />
<br />
* Started approximately 15 mL E coli B liquid culture overnight.<br />
<br />
* Worked on data analysis / picture processing for [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size|8.16 Modeling phage plaque size - Experiment One]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/8/13''' </font><br />
<br />
JL<br />
<br />
* Continued data analysis / picture processing for [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size|8.16 Modeling phage plaque size - Experiment One]].<br />
<br />
* Performed preliminary titer in preparation for repeating the modeling experiment with T1 and T2. For specifics, please see [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size|8.16 Modeling phage plaque size - Experiment One]].<br />
<br />
* Started approximately 25mL of E coli B liquid culture overnight<br />
<br />
<br><br />
<br />
<font size="4"> '''9/9/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed analysis of modeling result using ImageJ.<br />
<br />
* Repeated the modeling procedure in [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size|8.16 Modeling phage plaque size - Experiment One]] for T1 and T2. Incubation started at 4:30pm.<br />
<br />
* Started a fresh round for phage propagation for the Phage Purification Team. Specifically, to a 15mL centrifuge tube we added 8mL of LB, 2mL of E coli B liquid culture overnight, and 20uL of 8.24 T7 phage stock.<br />
<br />
* Divided up assignments for updating the wiki.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/10/13''' </font><br />
<br />
JL, LP<br />
<br />
* Took the T1 and T2 modeling plates our the incubation at 4:30pm.<br />
<br />
* Started approximately 25mL of E coli B liquid culture overnight. <br />
<br />
* Measured plaque sizes using ImageJ.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/11/13''' </font><br />
<br />
JL, LP<br />
<br />
* Isolated the phage (from 9.9) by centrifuging down the bacteria, transferring the supernatant to a new 15mL centrifuge tube, and adding 900ul of chloroform to the supernatant.<br />
<br />
* Took pictures of T1 and T2 plates for [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size|8.16 Modeling phage plaque size - Experiment One]].<br />
<br><br />
<br />
<font size="4"> '''9/12/13''' </font><br />
<br />
JL<br />
<br />
* Spot test showed plaques up to -7. This indicate that the phage suspension solution we gave the Phage Purification Team for CsCl was at least 10E9 pfu/mL.<br />
<br />
* Started approximately 30mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/13/13''' </font><br />
<br />
JL, LP<br />
<br />
* The Phage Purification Team has good news for us. They were able to observe banding in their latest CsCl gradient for T7. We thus decided to perform a new round of mutagenesis this weekend and hopefully selection next week.<br />
<br />
* In preparation for mutagenesis tomorrow, we performed titers for 8.24 T7 stock.<br />
<br />
* Started approximately 5mL of E coli B liquid culture overnight.<br />
<br />
* Streaked out E coli B.<br />
<br />
<font size="4"> '''9/14/13''' </font><br />
<br />
JL<br />
<br />
* Autoclaved ddH<sub>2</sub>O, LB, x2 top agar.<br />
<br />
* Used the autoclaved ddH<sub>2</sub>O to make adenine and uracil solution. Specifically,<br />
<br />
: - 254mg of uracil was dissolved in 10mL of ddH<sub>2</sub>O to produce a uracil solution with a concentration of approximately 2.5mg/mL<br />
<br />
: - 753mg of adenine hemisulfate dihydrate was dissolved in 10mL of ddH<sub>2</sub>O to produce a adenine solution with a concentration of approximately 5mg/mL<br />
<br />
* Started approximately 5mL of E coli B liquid culture overnight.<br />
<br />
<font size="4"> '''9/15/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed the mutagenesis procedure in [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]]<br />
<br />
* Started approximately 20mL of E coli B liquid culture overnight<br />
<br />
* Started phage propagation<br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period3/Dailylog2013-10-09T21:37:08Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: October 1 - October 15 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''9/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed plans for these future two weeks: established priority.<br />
<br />
* Help Large Phage Group with dilution series and performing a spot test for their small phage band. <br />
<br />
<br><br />
<br />
<font size="4"> '''9/17/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed CsCl gradient set up with Phage Purification Team.<br />
<br />
* Performed dilution series and spot test for samples post CsCl from [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/19/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/20/13''' </font><br />
<br />
LP<br />
<br />
* Performed "Plating to Check for Plaque Size" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/23/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Plating to Check for Plaque Size 2" and "Checking for Plaque Viability from Step 5" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/24/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 30 mL E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/25/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Checking for Plaque Viability 2" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Started approximately 25mL of E coli B liquid culture overnight<br />
<br />
<br><br />
<br />
<font size="4"> '''9/26/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 25mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/27/13''' </font><br />
<br />
JL, LP<br />
<br />
* Determined results for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Finished updating the wiki.<br />
<br />
* Started approximately 30mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period3/Dailylog2013-10-09T21:35:57Z<p>Perry721: Created page with "{{TeamBYUProvo}} <br> {| width="100%" | colspan="3" | <font color="#333399" size="5" font face="Calibri"> : '''Small Phage September - October Notebook: September 16 - Septem..."</p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: September 16 - September 30 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''9/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed plans for these future two weeks: established priority.<br />
<br />
* Help Large Phage Group with dilution series and performing a spot test for their small phage band. <br />
<br />
<br><br />
<br />
<font size="4"> '''9/17/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed CsCl gradient set up with Phage Purification Team.<br />
<br />
* Performed dilution series and spot test for samples post CsCl from [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/19/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/20/13''' </font><br />
<br />
LP<br />
<br />
* Performed "Plating to Check for Plaque Size" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/23/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Plating to Check for Plaque Size 2" and "Checking for Plaque Viability from Step 5" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/24/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 30 mL E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/25/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Checking for Plaque Viability 2" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Started approximately 25mL of E coli B liquid culture overnight<br />
<br />
<br><br />
<br />
<font size="4"> '''9/26/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 25mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/27/13''' </font><br />
<br />
JL, LP<br />
<br />
* Determined results for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Finished updating the wiki.<br />
<br />
* Started approximately 30mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/FallexpTeam:BYU Provo/Notebook/SmallPhage/Fallexp2013-10-09T21:34:36Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 50%; background-color: transparent;"|<br />
<br />
<font size="5" font face="Calibri"> '''September 1 - September 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> While the phage purification team was working out a kink in their cesium chloride gradient, we focused on modeling the relationship between capsid size and plaque size. Then once the cesium chloride gradient was perfected, we performed another round of mutagenesis, hoping that we would now be able to see mutant T7 bacteriophage. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''September 16 - September 30''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> All our hard work finally paid off! We managed to isolate a smaller and a larger T7 bacteriophage! </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period2/Dailylog|Daily log]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:BYUSPFall2.JPG|220px|center|link=]]<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''October 1 - October 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> Ad description. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''October 16 - October 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> Ad description. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period4/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/FallexpTeam:BYU Provo/Notebook/SmallPhage/Fallexp2013-10-09T21:33:56Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 50%; background-color: transparent;"|<br />
<br />
<font size="5" font face="Calibri"> '''September 1 - September 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> While the phage purification team was working out a kink in their cesium chloride gradient, we focused on modeling the relationship between capsid size and plaque size. Then once the cesium chloride gradient was perfected, we performed another round of mutagenesis, hoping that we would now be able to see mutant T7 bacteriophage. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''September 16 - September 30''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> All our hard work finally paid off! We managed to isolate a smaller and a larger T7 bacteriophage! </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
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[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period3/Dailylog|Daily log]]<br />
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{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/AttributionTeam:BYU Provo/Attribution2013-09-28T03:40:34Z<p>Perry721: </p>
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__NOTOC__==Attributions==<br />
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The undergraduate students of the BYU 2013 iGEM Team are actively involved in the invention, design, and implementation of our project, under the guidance of [http://lifesciences.byu.edu/~grosej Dr. Julianne Grose]. We are responsible for all the lab work, modeling, data analysis, wiki design, poster creation, and presentation development. This Phage Pharming project belongs to the entire team.<br />
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<br><br />
<br><br />
<br />
==Acknowledgements==<br />
<br />
===Brigham Young University Support===<br />
<br />
[[File:BYULifeSciences.JPG|130px|right|link=http://lifesciences.byu.edu/]]<br />
BYU 2013 iGEM TEAM would like to thank and acknowledge support from [http://lifesciences.byu.edu/ the College of Life Sciences] and [http://mmbio.byu.edu/ the Department of Microbiology & Molecular Biology] in terms of funding, support personnel, facilities, and equipments which made our project possible. Additionally, we are appreciative of all other [[Team:BYU_Provo/Attribution#Support_from_Additional_Professors_and_Research_Groups|departments and faculties]] who contributed to our project.<br />
<br />
<br><br />
<br />
===Professors and Teaching Assistant===<br />
All research work is performed by the undergraduate students on our team. Our professors and advisors provides support and suggestions to facilitate the implement of our project. We would like to thank the tireless contributions of our advisor [http://lifesciences.byu.edu/~grosej Dr. Julianne Grose]. Her dedication to our team and iGEM is indispensable to our overall success. And thanks to [http://lifesciences.byu.edu/~dlk5 Dr. David Kooyman] and Dr. Paul Price for their valuable insights.<br />
<br />
We recognize the work of our four devote graduate teaching assistants: Jordan Mackay, Whitney Hayes, Desi DeMille, and Bryan Badal. We are grateful for their assistance in the lab and the timely preparation of materials.<br />
<br />
<br><br />
<br />
===Support from Additional Professors and Research Groups===<br />
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We would like to thank the following group and individuals (listed in alphabetical order of last names) for their help with our project:<br />
<br />
* The [https://2012.igem.org/Team:Lyon-INSA Lyon-INSA iGEM Team] for a plasmid containing dispersin.<br />
<br />
* [http://www.path.utah.edu/research/cbi/sherwood-casjens/ Dr. Sherwood Casjens] (University of Utah Department of Pathology) for T4 phage and helpful insights. <br />
<br />
* [http://lifesciences.byu.edu/~drjbio Dr. Jamie Jensen] (BYU Department of Biology) for her helpful critique of the children's book and accompanying pre and post test. <br />
<br />
* [http://lifesciences.byu.edu/~wrm2 Dr. William McCleary] (BYU Department of Microbiology and Molecular Biology) for training and the use of the ultracentrifuge.<br />
<br />
* [http://www.biosci.utexas.edu/mgm/People/Faculty/profiles/?id=1707 Dr. Ian Molineux] (University of Texas at Austin Molecular Genetics and Microbiology Program) for T7 phage.<br />
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* [http://lifesciences.byu.edu/~rar5 Dr. Richard Robison] (BYU Department of Microbiology & Molecular Biology) for training on the safe handling and disposal of ''Vibrio cholerae'' and ''Bacillus subtilis''.<br />
<br />
* [http://biosci3.ucdavis.edu/FacultyAndResearch/FacultyProfile.aspx?FacultyID=215 Dr. John Roth] (UC Davis Department of Microbiology and Molecular Genetics) for ''E. coli'' strains containing Bacteriophage Lambda. <br />
<br />
* Dr. Michael Standing ([http://microscopy.byu.edu/ BYU Microscopy Lab]) for operating the transmission electron microscopy.<br />
<br />
* [http://www.bnl.gov/biosciences/staff/Studier.php Dr. F. William Studier] (Brookhaven National Laboratory) for valuable suggestions regarding our bacteriophage T7 experimental design.<br />
<br />
* [http://creativeworks.byu.edu/infocenter/tata.htm Dr. Giovanni Tata] (BYU Creative Works Office) for insights into publishing our children's book: [[Team:BYU_Provo/Outreach/Baxtor#Different_Editions|''The Adventure of Baxter Bacteria'']].<br />
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===Art and Technical Support===<br />
<br />
We acknowledge the efforts of <br />
<br />
* Redge Ballard in illustrating our children's book, [[Team:BYU_Provo/Outreach/Baxtor|''The Adventure of Baxter Bacteria'']], and other drawings.<br />
* Andrew Aston in developing our wiki.<br />
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<font size="4" font face="Calibri"> '''Redge Ballard''' </font> <br />
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<font size="3" font face="Calibri"> Redge was born in Rexburg, Idaho and has 7 brothers and sisters, a sister-in-law and one niece. He currently resides in Provo, Utah and is attending Brigham Young University as a computer science major. He intends to emphasize his CS major in the animation field. Since he was a small child he has always loved drawing and art in general. His areas of interest range from pen and pencil to ceramics, to digital, to animation. </font><br />
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<br></div>Perry721http://2013.igem.org/Team:BYU_Provo/ResultsTeam:BYU Provo/Results2013-09-28T03:35:28Z<p>Perry721: </p>
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'''Phage Library'''<br />
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This summer, we developed a cesium chloride gradient protocol for isolating larger and smaller phage. Utilizing this gradient, we were able to isolate mutant T4 bacteriophages that have distinctively larger or smaller capsids and less variability in capsid size when compared to the wild type. Similarly, we were also able to isolate mutant T7 bacteriophages that have smaller and larger than average capsid sizes.<br />
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'''Cholera'''<br />
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To tackle the disease Cholera, we identified a protein, Amylase, that disrupt Cholera's biofilm formation. In addition, we demonstrated that ''E. coli'' is capable of sensing Cholera and hypothesize that it does so through its SdiA protein. In fact, Cholera can induce Lambda from lysogenic ''E. coli''.<br />
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{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/ResultsTeam:BYU Provo/Results2013-09-28T03:35:17Z<p>Perry721: </p>
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<div>{{TeamBYUProvo}}<br />
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[[File:BYUResults.JPG|965px|center|link=Team:BYU_Provo/Results/Experimental]]<br />
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<font color="#333399" size="5" font face="Calibri"> <center> Achievements </center></font><br />
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<font color="#333399" size="3" font face="Calibri"> <br />
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'''Phage Library'''<br />
<br />
This summer, we developed a cesium chloride gradient protocol for isolating larger and smaller phage. Utilizing this gradient, we were able to isolate mutant T4 bacteriophages that have distinctively larger or smaller capsids and less variability in capsid size when compared to the wild type. Similarly, we were also able to isolate mutant T7 bacteriophages that have smaller and larger than average capsid size.<br />
<br />
<br><br />
<br />
'''Cholera'''<br />
<br />
To tackle the disease Cholera, we identified a protein, Amylase, that disrupt Cholera's biofilm formation. In addition, we demonstrated that ''E. coli'' is capable of sensing Cholera and hypothesize that it does so through its SdiA protein. In fact, Cholera can induce Lambda from lysogenic ''E. coli''.<br />
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<font color="#333399" size="5" font face="Calibri"> <center>[[Team:BYU_Provo/Results/Judge|Judging Criteria]] </center></font><br />
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[[File:BYUJudgeIcon.JPG|200px|center|link=Team:BYU_Provo/Results/Judge]]<br />
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{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/ResultsTeam:BYU Provo/Results2013-09-28T03:34:46Z<p>Perry721: </p>
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[[File:BYUResults.JPG|965px|center|link=Team:BYU_Provo/Results/Experimental]]<br />
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<font color="#333399" size="5" font face="Calibri"> <center> Achievements </center></font><br />
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<font color="#333399" size="3" font face="Calibri"> <br />
<br />
'''Phage Library'''<br />
<br />
This summer, we developed a cesium chloride gradient protocol for isolating larger and smaller phage. Utilizing this gradient, we were able to isolate mutant T4 bacteriophages that have distinctively larger or smaller capsids and less variability in capsid size when compared to the wild type. Similarly, we were also able to isolate mutant T7 bacteriophages that have smaller than average capsid size.<br />
<br />
<br><br />
<br />
'''Cholera'''<br />
<br />
To tackle the disease Cholera, we identified a protein, Amylase, that disrupt Cholera's biofilm formation. In addition, we demonstrated that ''E. coli'' is capable of sensing Cholera and hypothesize that it does so through its SdiA protein. In fact, Cholera can induce Lambda from lysogenic ''E. coli''.<br />
<br />
</font><br />
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| style="width: 22%; background-color: transparent;"|<br />
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<font color="#333399" size="5" font face="Calibri"> <center>[[Team:BYU_Provo/Results/Judge|Judging Criteria]] </center></font><br />
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[[File:BYUJudgeIcon.JPG|200px|center|link=Team:BYU_Provo/Results/Judge]]<br />
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{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/TeamMemberTeam:BYU Provo/TeamMember2013-09-28T03:27:50Z<p>Perry721: </p>
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: '''2013 BYU iGEM Team'''</font><br />
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: [[Team:BYU_Provo/TeamMember#Team_Video|Team Video]]<br />
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: [[Team:BYU_Provo/TeamMember#Team_Members|Undergraduates]]<br />
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: [[Team:BYU_Provo/TeamMember#Mentors|Mentors]]<br />
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==Team Members==<br />
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<font color="#333399" size="4" font face="Calibri"> '''Amber Brown''' </font> <br />
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<font color="#333399" size="3" font face="Calibri"> Amber is not the typical lab junkie that you would imagine. Rather than spending a lot of time indoors, she loves getting out! Between all of her hiking, rafting, and slacklining she manages to squeeze in several hours research time in the lab for the BYU iGEM team. Amber one day hopes to become a professor of microbiology, a good fit for someone who is so dedicated to her work. Amber has enjoyed the research opportunities offered by iGEM and found it to be a valuable experience! </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Arick Christopher''' </font> <br />
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<font color="#333399" size="3" font face="Calibri"> Despite Arick’s creepy looking picture right here, he is actually a pretty cool cat. Growing up a redneck in Redding, California, he somehow joined the hipster phase when he moved to Medford, Oregon. While he is not studying exercise science, he is busy rafting, hiking, or playing Dungeons and Dragons. He aspires to one day become an oral surgeon and is apparently quite the cook (according to his girlfriend...). </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Clarice Harrison''' </font> <br />
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<font color="#333399" size="3" font face="Calibri"> Clarice lives a life torn between the two loves of her life: Russia and molecular biology. Self proclaimed 'gene junkie' and русская сестра, she finally achieved a merger of these two worlds when she spent a month at the University of Moscow on a biotechnology internship. After that she didn't know what to do with her life. So of course she is going to apply for grad school. In her spare time she enjoys sleeping with her purple pillows, purple blanket, and copy of Eugene Onegin. </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Kendall Kiser''' </font> <br />
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<font color="#333399" size="3" font face="Calibri"> Kendall Kiser is homozygous negative for the “get-PCR-right-the-first-time” gene. Labmates marvel that he was ever admitted to an institution of higher learning. He insists that he’s “like, way smart” and “if PCR were baseball, I’d go 1 for 20 and still make a million bucks, so there.” He sticks his tongue out like he’s three. This childishness is to blame for the entire children’s book text.<br />
<br />
By other descriptions, he’s a Spanish-speakin’, piano-playin’ soccer-strikin’ neuroscience nerd. Kendall’s left-handed talent for calligraphy is legendary, and entirely useless in an age of word processing software. </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Darren Lasko''' </font> <br />
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<font color="#333399" size="3" font face="Calibri"> Darren Lasko is from Reno, Nevada. He is majoring in Microbiology and wants to go to Medical School, because he thinks it's cool. Darren is always the person in the lab who picks up the slack and gets the job done. When everything goes wrong, he'll lighten the mood. Darren also enjoys watching the Bachelorette, and is a real ladies man! </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Xiuqi (Jade) Li''' </font> <br />
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<font color="#333399" size="3" font face="Calibri"> There are only three parts to Jade's schedule: in the lab, on her way to the lab, or thinking about the lab. What can you say? She is a total lab junkie. Between iGEM and a Cancer Research Fellowship, Jade spent more time in the lab than outside it this summer. Being cooped up all day in a small room that most students are not aware of its existence, Jade is disappointed that she missed her chance of absorbing as much Vitamin D as possible during the best part of the year. But at least she got to make something "explode" safely in a biology lab (see picture on the right). <br />
<br />
Jade challenges everyone to guess what she is holding in the picture. If you can get it right in one try, she will offer you a piece of the famous BYU mint truffles! </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Bryan Merrill''' </font> <br />
<br />
<font color="#333399" size="3" font face="Calibri"> Bryan is a bacteriophage aficionado. In addition to his phage research in iGEM, he works as a TA for the Phage Hunters class at BYU and does phage genomics research on the side. He is convinced that "phage" and "phun" begin with the same two letters.<br />
<br>Outside of the lab and school, you'll find him training for a half-marathon, tending his honey bees, gardening, and trying to impress his amazing wife. In a rare moment of free time, he is most likely watching a good movie, playing (or writing) music, or visiting family, or watching BYU sports. (Go Cougars!) </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Kelton Peck''' </font> <br />
<br />
<font color="#333399" size="3" font face="Calibri"> This biography is written in loving memory of Kelton Peck, whose remains were discovered one dreary morning on the top floor of the Widsoe lab building. Rigor mortis had eternally fixed his clutch around a pipet as he was loading his last gel. While living, Kelton loved playing the violin and ukulele, tearing up the soccer field, exploring the great outdoors, and spending time with a good book. Before his untimely death ended all worldly aspirations, he had hoped to pursue a career in specialized pharmacy and pharmaceutical research. </font><br />
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[[File:BYU_Kelton_Bio.JPG|190px|right|link=]]<br />
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<font color="#333399" size="4" font face="Calibri"> '''Lindsey Perry''' </font> <br />
<br />
<font color="#333399" size="3" font face="Calibri"> LJ (which stands for Lindsey Jackson Perry IV) is from Laguna Hills, California. He is a junior and studying Microbiology. LJ wishes that there was more time in the week as it's filled with iGEM lab, 17.5 credit hours worth of classes, all the homework that comes with them, a chemistry lab TA job, and volunteering at the hospital and a local hospice program. He is doing all these things in preparation for applying to Medical School next spring. In his rare free time, LJ likes to play hockey, go hiking, or practice the guitar. </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Nathan Robert Sabin''' </font> <br />
<br />
<font color="#333399" size="3" font face="Calibri"> Before coming to BYU and joining the iGEM team, Nathan's only experience with bio-engineering came from learning about Mewtwo in Pokemon: The Movie. Needless to say, he has come a long way. Nathan is a sophomore microbiology student, but more accurately a rough, tough, farmbody from deep in the heart of Texas. Despite growing up on a farm, he never learned how to milk a cow. After three more years at BYU Nathan has plans to do the cool thing and go to medical school. <br />
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[[File:BYU2013NathanSabin.jpg|190px|right]]<br />
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[[File:BYUMichaelSchellhous.jpg|190px|left|link=]]<br />
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<font color="#333399" size="4" font face="Calibri"> '''Michael Schellhous''' </font> <br />
<br />
<font color="#333399" size="3" font face="Calibri"> Not your typical southern California guy, Michael has never been surfing. However, he has seen several movies that involve surfing, so we're pretty sure he'd be an expert if he ever tried. When declaring his major, he couldn't decide between chemistry and biology, so he made the only logical choice and chose biochemistry. This happened to turn out to be his real passion and he has a special interest in proteomics and lipidomics. He loves reading, racquetball, and just about anything outside. </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Keltzie Westra''' </font> <br />
<br />
<font color="#333399" size="3" font face="Calibri"> Keltzie Westra is a Senior majoring in Medical Laboratory Science from Sacramento, California. When she's not in the lab she enjoys traveling and spending time with her family. Some of her favorite things to do are nap, eat lots of candy and vacation. While she is thrilled to be a newlywed, she is still having a hard time remembering her last name after getting married last July! </font><br />
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[[File:KeltzieWestra.JPG|190px|right|link=]]<br />
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==Mentors==<br />
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<font color="#333399" size="4" font face="Calibri"> '''Dr. Julianne Grose, Microbiology & Molecular Biology''' </font> <br />
<br />
<font color="#333399" size="3" font face="Calibri"> Julianne Grose is an assistant professor in the Department of Molecular Biology and Microbiology. She earned a PhD in the Department of Biology at the University of Utah, in the lab of the Distinguished Professor John Roth, a member of the National Academy of Sciences. Prior to coming to BYU she was a Postdoctoral Research Fellow in the lab of Jared Rutter as well as a Research Scientist for Bioenergenics, a small pharmaceutical company. Her studies have focused on the regulation of NAD(P) biosynthesis and glucose metabolism in Salmonella and the yeast S. cerevisiae. </font><br />
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<font color="#333399" size="4" font face="Calibri"> '''Dr. David Kooyman, Physiology & Developmental Biology''' </font> <br />
<br />
<font color="#333399" size="3" font face="Calibri"> David Kooyman is a professor in the Department of Physiology and Developmental Biology. He earned a PhD in a multi-disciplinary Molecular and Cellular Biology Program at Ohio University. Prior to coming to BYU in 1997, David worked as a Senior Scientist at DNX, a biotechnology company owned by Baxter International. Since coming to BYU he has served as a department chair and associate dean. For several years his research laboratory has been focused on studies involving molecular and cellular approaches to animal physiology and cartilage biology. He is the author of numerous manuscripts in peer-reviewed journals including the prestigious journals Science and Nature Medicine. He regularly includes undergraduate students in his research. Over the past two years, his publications have included 37 different undergraduates as co-authors. He has been invited to make numerous presentations at national and international meetings, three times as a plenary speaker and chaired the 3rd International Conference on Transgenic Animals. David’s reputation has enabled him to establish collaborations with senior scientists at universities and institutes around the world. </font><br />
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[[File:BYUDrKooyman.JPG|190px|right]]<br />
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<br><br />
<br></div>Perry721http://2013.igem.org/File:AngelsLanding.JPGFile:AngelsLanding.JPG2013-09-28T03:26:49Z<p>Perry721: </p>
<hr />
<div></div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period2/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period2/Dailylog2013-09-28T03:16:31Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: September 16 - September 30 Daily Log'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
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<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''9/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed plans for these future two weeks: established priority.<br />
<br />
* Help Large Phage Group with dilution series and performing a spot test for their small phage band. <br />
<br />
<br><br />
<br />
<font size="4"> '''9/17/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed CsCl gradient set up with Phage Purification Team.<br />
<br />
* Performed dilution series and spot test for samples post CsCl from [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/19/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/20/13''' </font><br />
<br />
LP<br />
<br />
* Performed "Plating to Check for Plaque Size" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/23/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Plating to Check for Plaque Size 2" and "Checking for Plaque Viability from Step 5" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/24/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 30 mL E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/25/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Checking for Plaque Viability 2" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Started approximately 25mL of E coli B liquid culture overnight<br />
<br />
<br><br />
<br />
<font size="4"> '''9/26/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 25mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/27/13''' </font><br />
<br />
JL, LP<br />
<br />
* Determined results for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Finished updating the wiki.<br />
<br />
* Started approximately 30mL of E. coli B overnight.<br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period2/DailylogTeam:BYU Provo/Notebook/SmallPhage/Fallexp/Period2/Dailylog2013-09-28T03:12:31Z<p>Perry721: </p>
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<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook: September 16 - September 30 Daily Log'''</font><br />
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|- valign="top"<br />
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<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 82%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
<br />
<font size="4"> '''9/16/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed plans for these future two weeks: established priority.<br />
<br />
* Help Large Phage Group with dilution series and performing a spot test for their small phage band. <br />
<br />
<br><br />
<br />
<font size="4"> '''9/17/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/18/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed CsCl gradient set up with Phage Purification Team.<br />
<br />
* Performed dilution series and spot test for samples post CsCl from [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/19/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 20 mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/20/13''' </font><br />
<br />
LP<br />
<br />
* Performed "Plating to Check for Plaque Size" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/23/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Plating to Check for Plaque Size 2" and "Checking for Plaque Viability from Step 5" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
<br><br />
<br />
<font size="4"> '''9/24/13''' </font><br />
<br />
LP<br />
<br />
* Started approximately 30 mL E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/25/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed "Checking for Plaque Viability 2" for [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]].<br />
<br />
* Started approximately 25mL of E coli B liquid culture overnight<br />
<br />
<br><br />
<br />
<font size="4"> '''9/9/13''' </font><br />
<br />
JL, LP<br />
<br />
* Discussed analysis of modeling result using ImageJ.<br />
<br />
* Repeated the modeling procedure in [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size|8.16 Modeling phage plaque size - Experiment One]] for T1 and T2. Incubation started at 4:30pm.<br />
<br />
* Started a fresh round for phage propagation for the Phage Purification Team. Specifically, to a 15mL centrifuge tube we added 8mL of LB, 2mL of E coli B liquid culture overnight, and 20uL of 8.24 T7 phage stock.<br />
<br />
* Divided up assignments for updating the wiki.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/10/13''' </font><br />
<br />
JL, LP<br />
<br />
* Took the T1 and T2 modeling plates our the incubation at 4:30pm.<br />
<br />
* Started approximately 25mL of E coli B liquid culture overnight. <br />
<br />
* Measured plaque sizes using ImageJ.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/11/13''' </font><br />
<br />
JL, LP<br />
<br />
* Isolated the phage (from 9.9) by centrifuging down the bacteria, transferring the supernatant to a new 15mL centrifuge tube, and adding 900ul of chloroform to the supernatant.<br />
<br />
* Took pictures of T1 and T2 plates for [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size|8.16 Modeling phage plaque size - Experiment One]].<br />
<br><br />
<br />
<font size="4"> '''9/12/13''' </font><br />
<br />
JL<br />
<br />
* Spot test showed plaques up to -7. This indicate that the phage suspension solution we gave the Phage Purification Team for CsCl was at least 10E9 pfu/mL.<br />
<br />
* Started approximately 30mL of E coli B liquid culture overnight.<br />
<br />
<br><br />
<br />
<font size="4"> '''9/13/13''' </font><br />
<br />
JL, LP<br />
<br />
* The Phage Purification Team has good news for us. They were able to observe banding in their latest CsCl gradient for T7. We thus decided to perform a new round of mutagenesis this weekend and hopefully selection next week.<br />
<br />
* In preparation for mutagenesis tomorrow, we performed titers for 8.24 T7 stock.<br />
<br />
* Started approximately 5mL of E coli B liquid culture overnight.<br />
<br />
* Streaked out E coli B.<br />
<br />
<font size="4"> '''9/14/13''' </font><br />
<br />
JL<br />
<br />
* Autoclaved ddH<sub>2</sub>O, LB, x2 top agar.<br />
<br />
* Used the autoclaved ddH<sub>2</sub>O to make adenine and uracil solution. Specifically,<br />
<br />
: - 254mg of uracil was dissolved in 10mL of ddH<sub>2</sub>O to produce a uracil solution with a concentration of approximately 2.5mg/mL<br />
<br />
: - 753mg of adenine hemisulfate dihydrate was dissolved in 10mL of ddH<sub>2</sub>O to produce a adenine solution with a concentration of approximately 5mg/mL<br />
<br />
* Started approximately 5mL of E coli B liquid culture overnight.<br />
<br />
<font size="4"> '''9/15/13''' </font><br />
<br />
JL, LP<br />
<br />
* Performed the mutagenesis procedure in [[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/9.13_Mutagen_Concentration_Test|9.13 Mutagen Concentration Test - Ninth Protocol]]<br />
<br />
* Started approximately 20mL of E coli B liquid culture overnight<br />
<br />
* Started phage propagation<br />
<br />
<br><br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Results/ExperimentalTeam:BYU Provo/Results/Experimental2013-09-28T03:07:06Z<p>Perry721: </p>
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<div>{{TeamBYUProvo}}<br />
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<br><br />
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: '''Experimental Results'''</font><br />
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<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Results|Results Overview]]<br />
<br />
: [[Team:BYU_Provo/Results/Judge|Judging Criteria]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Bronze|Bronze]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Silver|Silver]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Gold|Gold]]<br />
<br />
: [[Team:BYU_Provo/Results/Experimental|Experimental Results]]<br />
<br />
: [[Team:BYU_Provo/Results/Modeling|Modeling Results]]<br />
<br />
: [[Team:BYU_Provo/Parts|Parts Submitted]]<br />
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</font><br />
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__NOTOC__=Phage Team=<br />
<br />
<br />
===Isolation of Phage Library===<br />
[[File:Changethecapsidsize.png|550px|center|link=https://static.igem.org/mediawiki/2013/3/36/Changethecapsidsize.png]]<br />
<br><br />
<br />
===Results of Mutant T4 Phage Isolation=== <br />
<br />
<br />
[[File:T4capsidvariation.png|550px|center|link=https://static.igem.org/mediawiki/2013/4/4e/T4capsidvariation.png]]<br />
<br />
<br />
[[File:T4CsClTube.png|550px|center|link=https://static.igem.org/mediawiki/2013/9/98/T4CsClTube.png]]<br />
<br><br />
<br />
===Results of Mutant T7 Phage Isolation===<br />
<br />
<br />
[[File:T7protocol.png|550px|center|link=https://static.igem.org/mediawiki/2013/6/62/T7protocol.png]]<br />
<br />
As part of creating our bacteriophage library, we wanted to isolate both smaller and larger T7 bacteriophages. To do so, we first mutagenized T7 by growing it in the presence of 5-bromodeoxyuridine, a mutagenic base analog. This produced many mutant T7 bacteriophages, but they were hard to find amongst all the wild type T7. Therefore the phage purification team ran the mutagenized bacteriophage through a cesium chloride gradient. This separated the phage according to size, with the biggest phage traveling farther down. After determining where the mutant phage was in the gradient, we then plated the phage and looked for plaques that were smaller or larger than normal. Out of the 31 plaques we selected, two of them reproduced their plaque sizes, revealing that they were indeed mutant T7 bacteriophage! This can be seen in the two pictures below. The S4 plate with small phage produced larger plaques that were 0.592cm on average in diameter, while the L8 plate with large phage produced smaller plaques that were 0.264cm on average in diameter (Wild type T7 made plaques 0.388cm on average in diameter). The next step will be to get pictures of the phage with an electron microscope and sequence the genome to determine where the mutations occurred.<br />
<br />
[[File:SmallResults.png|550px|center|link=https://static.igem.org/mediawiki/2013/f/fb/SmallResults.png]]<br />
<br />
[[File:LargeResults.png|550px|center|link=https://static.igem.org/mediawiki/2013/8/81/LargeResults.png]]<br />
<br><br />
<br />
<br><br />
<br />
=Cholera Team=<br />
<br />
===Cholera Induces Bacteriophage Lambda From Lysogeny to its Lytic Cycle===<br />
<br />
[[File:byuk.jpg|250px|right|link=]]<br />
<br />
We discovered that lysogenic bacteriophage lambda responds to ''V. cholerae'' by lysing its host. The picture to the right is of ''E.coli'' with integrated lambda prophage, plated as a top agar lawn. To the top plate we streaked cholera in the center, while the bottom plate had no cholera.<br />
<br />
We hypothesize that SdiA, a transcriptional activator in ''E.Coli'', senses cholera's autoinducer molecules. To test this, we have infected SdiA wild type and SdiA-knockout ''E.coli'' with bacteriophage lambda, and are in the process of performing top agar plaque-assay tests to confirm that lambda induction is SdiA-dependent. This image is in preparation.<br />
<br />
Furthermore, we are researching an alternative method for lambda induction. Two proteins, cI and cro, are pivotal in determining whether lambda assumes, respectively, a lysogenic or lytic state. We hypothesize that overexpression of the cro protein will influence lambda into its lytic cycle. We have cloned CRO behind an arabinose-inducible promoter, transformed the plasmid into ''E.coli'', and infected with lambda. We will plate top agar lawns of this strain with and without arabinose, and anticipate results within the week. This image is in preparation.<br />
<br />
<br><br />
<br />
===Designing SdiA to be Specific for Cholera===<br />
<br />
[[File:BYUK Apr10-4.JPG|250px|left|link=]]<br />
<br />
In the beginning our plan was to transform into E.coli the most essential parts of the ''V.cholerae'' quorum sensing system. This was done, but our response proteins GFP and RFP did not work as expected.Shown above are colonies that were not supposed to be glowing. So we decided to try mutating the quorum sensing system ''E.coli'' already has such that it senses only CAI-1 which is an auto-inducer specific to ''V.cholerae''. The following is a schematic of our approach.<br />
<br />
<br />
[[File:BYU slide 4 again.jpg|600px|center|link=]]<br />
<br />
Below is a T coffee alignment of SdiA (''E.coli'' protein involved in quorum sensing response) and CqsS (receptor protein in V.cholerae that can only sense CAI-1). It was found that the amino acids corresponding to the binding pocket for auto-inducers of both proteins lined up in the alignment indicating a similar structure. We hypothesized that if the five amino acids in SdiA that are known to be important in autoinducer molecule binding were changed we could make SdiA a more specific receptor instead of a promiscuous one. According to our alignment two of the five amino acids were already the same between SdiA and CqsS (black arrows). The other three amino acids we decided to change in SdiA through site directed mutagenesis to be the same as CqsS (blue arrows). <br />
<br />
[[File:BYU CqsS SdiA binding pocket.png|600px|center|link=]]<br />
[[File:BYU planned experiment SdiAmutant.jpg|600px|center|link=]]<br />
<br />
<br><br />
<br />
===Biofilm inhibition by Amylase===<br />
<br />
The enzymatic activity of α-Amylase was characterized to determine its capacity to inhibit biofilm formation by ''V. cholerae''. Samples were prepared by adding 50 µL of ''V. cholerae'' culture to 1 mL of a high salt LB. The samples were then treated by the addition of purified α-Amylase in various concentrations and allowed to incubate at 30°C for 48 hours. After 48 hours the samples were examined and a distinct difference was seen in the amount of biofilm formed in the treated and untreated samples.<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssay1.jpg|450 px|link=https://static.igem.org/mediawiki/2013/2/2e/BYU2013-BiofilmAssay1.jpg]] [[File:BYU2013-BiofilmAssay2.JPG|249 px|link=https://static.igem.org/mediawiki/2013/e/ea/BYU2013-BiofilmAssay2.JPG]]</div><br />
<br />
<br />
The samples were then transferred to eppendorf tubes and centrifuged at 16,000 × g for two minutes, the supernatant was discarded to remove the growth media, and the samples were resuspended in 200 µL ddH2O. The samples were then stained with 50 µL of a 0.03% CV solution and allowed to incubate for five minutes. The samples were again centrifuged at 16,000 × g for two minutes and the supernatant containing excess CV was discarded. The pelleted biofilm was then washed with 800 µL of 95% EtOH without resuspension, centrifuged for 30 seconds, and the EtOH was discarded. The EtOH wash was repeated twice more for a total of three washes. The samples were then resuspended in 200 µL EtOH, transferred to a 96-well plate, and incubated for five minutes. The plate was then shaken for ten seconds and absorbance readings were taken for each sample at 540 nm, the absorbance wavelength of CV.<br />
<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssayGraph1.jpg|link=https://static.igem.org/mediawiki/2013/2/21/BYU2013-BiofilmAssayGraph1.jpg]]</div><br />
<br />
The above graph shows both the average pellet weight and the average absorbance readings for samples treated with 0, 2.5, 12.5, and 25 µL of α-Amylase. There is a distinct reduction in the amount of biofilm growth between untreated and treated samples, with samples treated with 25 µL α-Amylase showing a 65.8% decrease in biofilm formation after 48 hours. While this clearly shows the ability of α-Amylase to inhibit biofilm formation by ''V. cholerae'', further characterization is needed to determine the capacity of α-Amylase to degrade preexisting biofilms.<br />
<br />
</font><br />
<br><br />
<br />
<br><br />
<br />
<br></div>Perry721http://2013.igem.org/Team:BYU_Provo/Results/ExperimentalTeam:BYU Provo/Results/Experimental2013-09-28T03:04:41Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''Experimental Results'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Results|Results Overview]]<br />
<br />
: [[Team:BYU_Provo/Results/Judge|Judging Criteria]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Bronze|Bronze]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Silver|Silver]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Gold|Gold]]<br />
<br />
: [[Team:BYU_Provo/Results/Experimental|Experimental Results]]<br />
<br />
: [[Team:BYU_Provo/Results/Modeling|Modeling Results]]<br />
<br />
: [[Team:BYU_Provo/Parts|Parts Submitted]]<br />
<br />
</font><br />
<br />
| style="width: 81%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__=Phage Team=<br />
<br />
<br />
===Isolation of Phage Library===<br />
[[File:Changethecapsidsize.png|550px|center|link=https://static.igem.org/mediawiki/2013/3/36/Changethecapsidsize.png]]<br />
<br><br />
<br />
===Results of Mutant T4 Phage Isolation=== <br />
<br />
<br />
[[File:T4capsidvariation.png|550px|center|link=https://static.igem.org/mediawiki/2013/4/4e/T4capsidvariation.png]]<br />
<br />
<br />
[[File:T4CsClTube.png|550px|center|link=https://static.igem.org/mediawiki/2013/9/98/T4CsClTube.png]]<br />
<br><br />
<br />
===Results of Mutant T7 Phage Isolation===<br />
<br />
<br />
[[File:T7protocol.png|550px|center|link=https://static.igem.org/mediawiki/2013/6/62/T7protocol.png]]<br />
<br />
As part of creating our bacteriophage library, we wanted to isolate both smaller and larger T7 bacteriophages. To do so, we first mutagenized T7 by growing it in the presence of 5-bromodeoxyuridine, a mutagenic base analog. This produced many mutant T7 bacteriophages, but they were hard to find amongst all the wild type T7. Therefore the phage purification team ran the mutagenized bacteriophage through a cesium chloride gradient. This separated the phage according to size, with the biggest phage traveling farther down. After determining where the mutant phage is in the gradient, we then plated the phage and looked for plaques that were smaller or larger than normal. Out of the 31 plaques we selected, two of them reproduced their plaque sizes, revealing that they were indeed mutant T7 bacteriophage! This can be seen in the two pictures below. The S4 plate with small phage produced larger plaques that were 0.592cm on average in diameter, while the L8 plate with large phage produced smaller plaques that were 0.264cm on average in diameter (Wild type T7 made plaques 0.388cm on average in diameter). The next step will be to get pictures of the phage with an electron microscope and sequence the genome to determine where the mutations occurred.<br />
<br />
[[File:SmallResults.png|550px|center|link=https://static.igem.org/mediawiki/2013/f/fb/SmallResults.png]]<br />
<br />
[[File:LargeResults.png|550px|center|link=https://static.igem.org/mediawiki/2013/8/81/LargeResults.png]]<br />
<br><br />
<br />
<br><br />
<br />
=Cholera Team=<br />
<br />
===Cholera Induces Bacteriophage Lambda From Lysogeny to its Lytic Cycle===<br />
<br />
[[File:byuk.jpg|250px|right|link=]]<br />
<br />
We discovered that lysogenic bacteriophage lambda responds to ''V. cholerae'' by lysing its host. The picture to the right is of ''E.coli'' with integrated lambda prophage, plated as a top agar lawn. To the top plate we streaked cholera in the center, while the bottom plate had no cholera.<br />
<br />
We hypothesize that SdiA, a transcriptional activator in ''E.Coli'', senses cholera's autoinducer molecules. To test this, we have infected SdiA wild type and SdiA-knockout ''E.coli'' with bacteriophage lambda, and are in the process of performing top agar plaque-assay tests to confirm that lambda induction is SdiA-dependent. This image is in preparation.<br />
<br />
Furthermore, we are researching an alternative method for lambda induction. Two proteins, cI and cro, are pivotal in determining whether lambda assumes, respectively, a lysogenic or lytic state. We hypothesize that overexpression of the cro protein will influence lambda into its lytic cycle. We have cloned CRO behind an arabinose-inducible promoter, transformed the plasmid into ''E.coli'', and infected with lambda. We will plate top agar lawns of this strain with and without arabinose, and anticipate results within the week. This image is in preparation.<br />
<br />
<br><br />
<br />
===Designing SdiA to be Specific for Cholera===<br />
<br />
[[File:BYUK Apr10-4.JPG|250px|left|link=]]<br />
<br />
In the beginning our plan was to transform into E.coli the most essential parts of the ''V.cholerae'' quorum sensing system. This was done, but our response proteins GFP and RFP did not work as expected.Shown above are colonies that were not supposed to be glowing. So we decided to try mutating the quorum sensing system ''E.coli'' already has such that it senses only CAI-1 which is an auto-inducer specific to ''V.cholerae''. The following is a schematic of our approach.<br />
<br />
<br />
[[File:BYU slide 4 again.jpg|600px|center|link=]]<br />
<br />
Below is a T coffee alignment of SdiA (''E.coli'' protein involved in quorum sensing response) and CqsS (receptor protein in V.cholerae that can only sense CAI-1). It was found that the amino acids corresponding to the binding pocket for auto-inducers of both proteins lined up in the alignment indicating a similar structure. We hypothesized that if the five amino acids in SdiA that are known to be important in autoinducer molecule binding were changed we could make SdiA a more specific receptor instead of a promiscuous one. According to our alignment two of the five amino acids were already the same between SdiA and CqsS (black arrows). The other three amino acids we decided to change in SdiA through site directed mutagenesis to be the same as CqsS (blue arrows). <br />
<br />
[[File:BYU CqsS SdiA binding pocket.png|600px|center|link=]]<br />
[[File:BYU planned experiment SdiAmutant.jpg|600px|center|link=]]<br />
<br />
<br><br />
<br />
===Biofilm inhibition by Amylase===<br />
<br />
The enzymatic activity of α-Amylase was characterized to determine its capacity to inhibit biofilm formation by ''V. cholerae''. Samples were prepared by adding 50 µL of ''V. cholerae'' culture to 1 mL of a high salt LB. The samples were then treated by the addition of purified α-Amylase in various concentrations and allowed to incubate at 30°C for 48 hours. After 48 hours the samples were examined and a distinct difference was seen in the amount of biofilm formed in the treated and untreated samples.<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssay1.jpg|450 px|link=https://static.igem.org/mediawiki/2013/2/2e/BYU2013-BiofilmAssay1.jpg]] [[File:BYU2013-BiofilmAssay2.JPG|249 px|link=https://static.igem.org/mediawiki/2013/e/ea/BYU2013-BiofilmAssay2.JPG]]</div><br />
<br />
<br />
The samples were then transferred to eppendorf tubes and centrifuged at 16,000 × g for two minutes, the supernatant was discarded to remove the growth media, and the samples were resuspended in 200 µL ddH2O. The samples were then stained with 50 µL of a 0.03% CV solution and allowed to incubate for five minutes. The samples were again centrifuged at 16,000 × g for two minutes and the supernatant containing excess CV was discarded. The pelleted biofilm was then washed with 800 µL of 95% EtOH without resuspension, centrifuged for 30 seconds, and the EtOH was discarded. The EtOH wash was repeated twice more for a total of three washes. The samples were then resuspended in 200 µL EtOH, transferred to a 96-well plate, and incubated for five minutes. The plate was then shaken for ten seconds and absorbance readings were taken for each sample at 540 nm, the absorbance wavelength of CV.<br />
<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssayGraph1.jpg|link=https://static.igem.org/mediawiki/2013/2/21/BYU2013-BiofilmAssayGraph1.jpg]]</div><br />
<br />
The above graph shows both the average pellet weight and the average absorbance readings for samples treated with 0, 2.5, 12.5, and 25 µL of α-Amylase. There is a distinct reduction in the amount of biofilm growth between untreated and treated samples, with samples treated with 25 µL α-Amylase showing a 65.8% decrease in biofilm formation after 48 hours. While this clearly shows the ability of α-Amylase to inhibit biofilm formation by ''V. cholerae'', further characterization is needed to determine the capacity of α-Amylase to degrade preexisting biofilms.<br />
<br />
</font><br />
<br><br />
<br />
<br><br />
<br />
<br></div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/SummerexpTeam:BYU Provo/Notebook/SmallPhage/Summerexp2013-09-28T02:42:06Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage July - August Notebook'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 50%; background-color: transparent;"|<br />
<br />
<font size="5" font face="Calibri"> '''July 1 - July 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> A new term and a fresh start! During these two weeks, we performed two more rounds of mutagenesis. With every experiment, we were able to perfect our protocol. Although our results are still not satisfactory, we are getting close! Eventually, we discovered that our phage stock decreased in titer during its preservation inside the fridge. We will propagate our phage and try again!</font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period1/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period1/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:lsadjf.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''July 16 - July 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> During these couple weeks, we tried another 2 rounds of mutagenesis. The 5th round didn't work well so we made some major adjustments for the 6th round of mutagenesis. We believe that we were successful in finally mutating the phage and are giving them to the phage purification team to run a cesium chloride gradient on them. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period2/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period2/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:ouawr.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''August 1 - August 16''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> Our goals for these two weeks were very clear. First, we need to generate enough mutagenized phage for the Phage Purification Team to work with. Second, we need to start our modeling project. Hopefully, everything will go according to plan and we will have presentable data soon! As for our modeling project, we have decide to model phage plaque sizes against different variables, such as phage particle size and top agar concentration. We aim to generate a mathematical model that can be used both to optimize phage plaque size for research and characterize our mutant phage. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period3/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period3/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:nsdfj.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''August 17 - August 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> These two weeks are the interim between summer term and fall semester at BYU. During these two weeks, we focused mostly on finalizing our mutagenesis procedure with the help of Phage Purification Team and their CsCl gradient protocol. We completed our seventh round of mutagenesis and we did see a variability in the size of phage plaques indication variability in phage capsid size. We conducted the eighth round of mutagenesis even more meticulously. Toward the end of these two-week period, we are working on characterizing phage. From our preliminary data, we are seeing the most concentrated phage after mutagenesis so far, which is very promising. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period4/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period4/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:6Small.JPG|220px|center]]<br />
<br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/SummerexpTeam:BYU Provo/Notebook/SmallPhage/Summerexp2013-09-28T02:41:18Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage July - August Notebook'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 50%; background-color: transparent;"|<br />
<br />
<font size="5" font face="Calibri"> '''July 1 - July 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> A new term and a fresh start! During these two weeks, we performed two more rounds of mutagenesis. With every experiment, we were able to perfect our protocol. Although our results are still not satisfactory, we are getting close! Eventually, we discovered that our phage stock decreased in titer during its preservation inside the fridge. We will propagate our phage and try again!</font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period1/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period1/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:lsadjf.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''July 16 - July 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> During these couple weeks, we tried another 2 rounds of mutagenesis. The 5th round didn't work well so we made some major adjustments for the 6th round of mutagenesis. We believe that we were successful in finally mutating the phage and are giving them to the phage purification team to run a cesium chloride gradient on them. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period2/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period2/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:ouawr.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''August 1 - August 16''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> Our goals for these two weeks were very clear. First, we need to generate enough mutagenized phage for the Phage Purification Team to work with. Second, we need to start our modeling project. Hopefully, everything will go according to plan and we will have presentable data soon! As for our modeling project, we have decide to model phage plaque sizes against different variables, such as phage particle size and top agar concentration. We aim to generate a mathematical model that can be used both to optimize phage plaque size for research and characterize our mutant phage. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period3/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period3/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:nsdfj.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''August 17 - August 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> These two weeks are the interim between summer term and fall semester at BYU. During these two weeks, we focused mostly on finalizing our mutagenesis procedure with the help of Phage Purification Team and their CsCl gradient protocol. We completed our seventh round of mutagenesis and we did see a variability in the size of phage plaques indication variability in phage capsid size. We conducted the eighth round of mutagenesis even more meticulously. Toward the end of these two-week period, we are working on characterizing phage. From our preliminary data, we are seeing the most concentrated phage after mutagenesis so far, which is very promising. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period4/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period4/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:6small.JPG|220px|center]]<br />
<br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/File:Nsdfj.JPGFile:Nsdfj.JPG2013-09-28T02:39:06Z<p>Perry721: </p>
<hr />
<div></div>Perry721http://2013.igem.org/File:Ouawr.JPGFile:Ouawr.JPG2013-09-28T02:38:49Z<p>Perry721: </p>
<hr />
<div></div>Perry721http://2013.igem.org/File:Lsadjf.JPGFile:Lsadjf.JPG2013-09-28T02:38:19Z<p>Perry721: </p>
<hr />
<div></div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/SummerexpTeam:BYU Provo/Notebook/SmallPhage/Summerexp2013-09-28T02:38:05Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage July - August Notebook'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 50%; background-color: transparent;"|<br />
<br />
<font size="5" font face="Calibri"> '''July 1 - July 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> A new term and a fresh start! During these two weeks, we performed two more rounds of mutagenesis. With every experiment, we were able to perfect our protocol. Although our results are still not satisfactory, we are getting close! Eventually, we discovered that our phage stock decreased in titer during its preservation inside the fridge. We will propagate our phage and try again!</font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period1/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period1/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:lsadjf.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''July 16 - July 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> During these couple weeks, we tried another 2 rounds of mutagenesis. The 5th round didn't work well so we made some major adjustments for the 6th round of mutagenesis. We believe that we were successful in finally mutating the phage and are giving them to the phage purification team to run a cesium chloride gradient on them. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period2/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period2/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:ouawr.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''August 1 - August 16''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> Our goals for these two weeks were very clear. First, we need to generate enough mutagenized phage for the Phage Purification Team to work with. Second, we need to start our modeling project. Hopefully, everything will go according to plan and we will have presentable data soon! As for our modeling project, we have decide to model phage plaque sizes against different variables, such as phage particle size and top agar concentration. We aim to generate a mathematical model that can be used both to optimize phage plaque size for research and characterize our mutant phage. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period3/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period3/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:nsdfj.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''August 17 - August 31''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> These two weeks are the interim between summer term and fall semester at BYU. During these two weeks, we focused mostly on finalizing our mutagenesis procedure with the help of Phage Purification Team and their CsCl gradient protocol. We completed our seventh round of mutagenesis and we did see a variability in the size of phage plaques indication variability in phage capsid size. We conducted the eighth round of mutagenesis even more meticulously. Toward the end of these two-week period, we are working on characterizing phage. From our preliminary data, we are seeing the most concentrated phage after mutagenesis so far, which is very promising. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period4/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Summerexp/Period4/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:8dhf.JPG|220px|center]]<br />
<br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/File:Asdfsa.JPGFile:Asdfsa.JPG2013-09-28T02:33:05Z<p>Perry721: </p>
<hr />
<div></div>Perry721http://2013.igem.org/Team:BYU_Provo/Notebook/SmallPhage/FallexpTeam:BYU Provo/Notebook/SmallPhage/Fallexp2013-09-28T02:32:40Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="3" | <font color="#333399" size="5" font face="Calibri"> <br />
<br />
: '''Small Phage September - October Notebook'''</font><br />
<br />
<br><br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
<font size = "4"><br />
<br />
: <u> '''Small Phage''' </u> </font><br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]<br />
<br />
: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]<br />
<br />
</font><br />
<br />
| style="width: 50%; background-color: transparent;"|<br />
<br />
<font size="5" font face="Calibri"> '''September 1 - September 15''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> While the phage purification team was working out a kink in their cesium chloride gradient, we focused on modeling the relationship between capsid size and plaque size. Then once the cesium chloride gradient was perfected, we performed another round of mutagenesis, hoping that we would now be able to see mutant T7 bacteriophage. </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Dailylog|Daily log]]<br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period1/Explist|Experiment Listing]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:asdfsa.JPG|220px|center]]<br />
<br />
<br />
|-<br />
<br />
| style="width: 18%; background-color: transparent;"|<br />
<br />
| <font size="5" font face="Calibri"> '''September 16 - September 30''' </font><br />
<br />
<br> <br />
<br />
<font size="3" font face="Calibri"> All our hard work finally paid off! We managed to isolate a smaller and a larger T7 bacteriophage! </font><br />
<br />
<br><br />
<br />
<font color="#333399" size="4" font face="Calibri"><br />
<br />
[[Team:BYU_Provo/Notebook/SmallPhage/Fallexp/Period2/Dailylog|Daily log]]<br />
<br />
</font><br />
<br />
<br><br />
<br />
<br />
| style="width: 20%; background-color: transparent;"| [[File:BYUSPFall2.JPG|220px|center|link=]]<br />
<br />
<br />
|}<br />
<br />
{{TeamBYUProvoFooter}}</div>Perry721http://2013.igem.org/Team:BYU_Provo/Results/ExperimentalTeam:BYU Provo/Results/Experimental2013-09-28T02:25:35Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''Experimental Results'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Results|Results Overview]]<br />
<br />
: [[Team:BYU_Provo/Results/Judge|Judging Criteria]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Bronze|Bronze]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Silver|Silver]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Gold|Gold]]<br />
<br />
: [[Team:BYU_Provo/Results/Experimental|Experimental Results]]<br />
<br />
: [[Team:BYU_Provo/Results/Modeling|Modeling Results]]<br />
<br />
: [[Team:BYU_Provo/Parts|Parts Submitted]]<br />
<br />
</font><br />
<br />
| style="width: 81%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Phage Team==<br />
<br />
<br />
===Isolation of Phage Library===<br />
[[File:Changethecapsidsize.png|600px|center|link=https://static.igem.org/mediawiki/2013/3/36/Changethecapsidsize.png]]<br />
<br><br />
<br />
===Results of Mutant T4 Phage Isolation=== <br />
<br />
<br />
[[File:T4capsidvariation.png|600px|center|link=https://static.igem.org/mediawiki/2013/4/4e/T4capsidvariation.png]]<br />
<br />
<br />
[[File:T4CsClTube.png|600px|center|link=https://static.igem.org/mediawiki/2013/9/98/T4CsClTube.png]]<br />
<br><br />
<br />
===Results of Mutant T7 Phage Isolation===<br />
<br />
<br />
[[File:T7protocol.png|600px|center|]]<br />
<br />
As part of creating our bacteriophage library, we wanted to isolate both smaller and larger T7 bacteriophages. To do so, we first mutagenized T7 by growing it in the presence of 5-bromodeoxyuridine, a mutagenic base analog. This produced many mutant T7 bacteriophages, but they were hard to find amongst all the wild type T7. Therefore the phage purification team ran the mutagenized bacteriophage through a cesium chloride gradient. This separated the phage according to size, with the biggest phage traveling farther down. After determining where the mutant phage is in the gradient, we then plated the phage and looked for plaques that were smaller or larger than normal. Out of the 31 plaques we selected, two of them reproduced their plaque sizes, revealing that they were indeed mutant T7 bacteriophage! This can be seen in the two pictures below. The S4 plate with small phage produced larger plaques, while the L8 plate with large phage produced smaller plaques. The next step will be to get pictures of the phage with an electron microscope and sequence the genome to determine where the mutations occurred.<br />
<br />
[[File:SmallResults.png|600px|center|]]<br />
<br />
[[File:LargeResults.png|600px|center|]]<br />
<br><br />
<br />
<br><br />
<br />
==Cholera Team==<br />
<br />
===Cholera Induces Bacteriophage Lambda From Lysogeny to its Lytic Cycle===<br />
<br />
[[File:byuk.jpg|250px|center|link=]]<br />
<br />
<br><br />
<br />
===Designing SdiA to be Specific for Cholera===<br />
<br />
[[File:BYUK Apr10-4.JPG|250px|center|link=]]<br />
<br />
In the beginning our plan was to transform into E.coli the most essential parts of the V.cholerae quorum sensing system. This was done, but our response proteins GFP and RFP did not work as expected.Shown above are colonies that were not supposed to be glowing. So we decided to try mutating the quorum sensing system E.coli already had to be able to only sense CAI-1 which is an auto-inducer specific to V.cholerae. <br />
<br />
[[File:BYU slide 4 again.jpg|600px|center|link=]]<br />
[[File:BYU CqsS SdiA binding pocket.png|600px|center|link=]]<br />
[[File:BYU planned experiment SdiAmutant.jpg|600px|center|link=]]<br />
<br />
<br><br />
<br />
===Biofilm inhibition by Amylase===<br />
<br />
The enzymatic activity of α-Amylase was characterized to determine its capacity to inhibit biofilm formation by ''V. cholerae''. Samples were prepared by adding 50 µL of ''V. cholerae'' culture to 1 mL of a high salt LB. The samples were then treated by the addition of purified α-Amylase in various concentrations and allowed to incubate at 30°C for 48 hours. After 48 hours the samples were examined and a distinct difference was seen in the amount of biofilm formed in the treated and untreated samples.<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssay1.jpg|450 px|link=https://static.igem.org/mediawiki/2013/2/2e/BYU2013-BiofilmAssay1.jpg]] [[File:BYU2013-BiofilmAssay2.JPG|249 px|link=https://static.igem.org/mediawiki/2013/e/ea/BYU2013-BiofilmAssay2.JPG]]</div><br />
<br />
<br />
The samples were then transferred to eppendorf tubes and centrifuged at 16,000 × g for two minutes, the supernatant was discarded to remove the growth media, and the samples were resuspended in 200 µL ddH2O. The samples were then stained with 50 µL of a 0.03% CV solution and allowed to incubate for five minutes. The samples were again centrifuged at 16,000 × g for two minutes and the supernatant containing excess CV was discarded. The pelleted biofilm was then washed with 800 µL of 95% EtOH without resuspension, centrifuged for 30 seconds, and the EtOH was discarded. The EtOH wash was repeated twice more for a total of three washes. The samples were then resuspended in 200 µL EtOH, transferred to a 96-well plate, and incubated for five minutes. The plate was then shaken for ten seconds and absorbance readings were taken for each sample at 540 nm, the absorbance wavelength of CV.<br />
<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssayGraph1.jpg|link=https://static.igem.org/mediawiki/2013/2/21/BYU2013-BiofilmAssayGraph1.jpg]]</div><br />
<br />
The above graph shows both the average pellet weight and the average absorbance readings for samples treated with 0, 2.5, 12.5, and 25 µL of α-Amylase. There is a distinct reduction in the amount of biofilm growth between untreated and treated samples, with samples treated with 25 µL α-Amylase showing a 65.8% decrease in biofilm formation after 48 hours. While this clearly shows the ability of α-Amylase to inhibit biofilm formation by ''V. cholerae'', further characterization is needed to determine the capacity of α-Amylase to degrade preexisting biofilms.<br />
<br />
</font><br />
<br><br />
<br />
<br><br />
<br />
<br></div>Perry721http://2013.igem.org/Team:BYU_Provo/Results/ExperimentalTeam:BYU Provo/Results/Experimental2013-09-28T02:22:21Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''Experimental Results'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Results|Results Overview]]<br />
<br />
: [[Team:BYU_Provo/Results/Judge|Judging Criteria]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Bronze|Bronze]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Silver|Silver]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Gold|Gold]]<br />
<br />
: [[Team:BYU_Provo/Results/Experimental|Experimental Results]]<br />
<br />
: [[Team:BYU_Provo/Results/Modeling|Modeling Results]]<br />
<br />
: [[Team:BYU_Provo/Parts|Parts Submitted]]<br />
<br />
</font><br />
<br />
| style="width: 81%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Phage Team==<br />
<br />
<br />
===Isolation of Phage Library===<br />
[[File:Changethecapsidsize.png|600px|center|link=https://static.igem.org/mediawiki/2013/3/36/Changethecapsidsize.png]]<br />
<br><br />
<br />
===Results of Mutant T4 Phage Isolation=== <br />
<br />
<br />
[[File:T4capsidvariation.png|600px|center|link=https://static.igem.org/mediawiki/2013/4/4e/T4capsidvariation.png]]<br />
<br />
<br />
[[File:T4CsClTube.png|600px|center|link=https://static.igem.org/mediawiki/2013/9/98/T4CsClTube.png]]<br />
<br><br />
<br />
===Results of Mutant T7 Phage Isolation===<br />
<br />
<br />
[[File:T7protocol.png|600px|center|]]<br />
<br />
As part of creating our bacteriophage library, we wanted to isolate both smaller and larger T7 bacteriophage. To do so, we first mutagenized T7 by growing it in the presence of 5-bromodeoxyuridine, a mutagenic base analog. This produces many mutant T7 bacteriophages, but they are hard to find amongst all the wild type T7. Therefore the phage purification team ran the mutagenized bacteriophage through a cesium chloride gradient. This separates the phage according to size, with the biggest traveling farther down. After determining where the mutant phage is in the gradient, we then plated the phage and looked for plaques that were smaller or larger than normal. Out of the 31 plaques we selected, two of them reproduced their plaque sizes, revealing that they were indeed mutant T7 bacteriophage! This can be seen in the two pictures below. The S4 plate with small phage produced larger plaques, while the L8 plate with large phage produced smaller plaques. The next step will be to get pictures of the phage with an electron microscope and sequence the genome to determine where the mutations occurred.<br />
<br />
[[File:SmallResults.png|600px|center|]]<br />
<br />
[[File:LargeResults.png|600px|center|]]<br />
<br><br />
<br />
<br><br />
<br />
==Cholera Team==<br />
<br />
===Cholera Induces Bacteriophage Lambda From Lysogeny to its Lytic Cycle===<br />
<br />
[[File:byuk.jpg|250px|center|link=]]<br />
<br />
<br><br />
<br />
===Designing SdiA to be Specific for Cholera===<br />
<br />
[[File:BYUK Apr10-4.JPG|250px|center|link=]]<br />
<br />
In the beginning our plan was to transform into E.coli the most essential parts of the V.cholerae quorum sensing system. This was done, but our response proteins GFP and RFP did not work as expected.Shown above are colonies that were not supposed to be glowing. So we decided to try mutating the quorum sensing system E.coli already had to be able to only sense CAI-1 which is an auto-inducer specific to V.cholerae. <br />
<br />
[[File:BYU slide 4 again.jpg|600px|center|link=]]<br />
[[File:BYU CqsS SdiA binding pocket.png|600px|center|link=]]<br />
<br><br />
<br />
===Biofilm inhibition by Amylase===<br />
<br />
The enzymatic activity of α-Amylase was characterized to determine its capacity to inhibit biofilm formation by ''V. cholerae''. Samples were prepared by adding 50 µL of ''V. cholerae'' culture to 1 mL of a high salt LB. The samples were then treated by the addition of purified α-Amylase in various concentrations and allowed to incubate at 30°C for 48 hours. After 48 hours the samples were examined and a distinct difference was seen in the amount of biofilm formed in the treated and untreated samples.<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssay1.jpg|450 px|link=https://static.igem.org/mediawiki/2013/2/2e/BYU2013-BiofilmAssay1.jpg]] [[File:BYU2013-BiofilmAssay2.JPG|249 px|link=https://static.igem.org/mediawiki/2013/e/ea/BYU2013-BiofilmAssay2.JPG]]</div><br />
<br />
<br />
The samples were then transferred to eppendorf tubes and centrifuged at 16,000 × g for two minutes, the supernatant was discarded to remove the growth media, and the samples were resuspended in 200 µL ddH2O. The samples were then stained with 50 µL of a 0.03% CV solution and allowed to incubate for five minutes. The samples were again centrifuged at 16,000 × g for two minutes and the supernatant containing excess CV was discarded. The pelleted biofilm was then washed with 800 µL of 95% EtOH without resuspension, centrifuged for 30 seconds, and the EtOH was discarded. The EtOH wash was repeated twice more for a total of three washes. The samples were then resuspended in 200 µL EtOH, transferred to a 96-well plate, and incubated for five minutes. The plate was then shaken for ten seconds and absorbance readings were taken for each sample at 540 nm, the absorbance wavelength of CV.<br />
<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssayGraph1.jpg|link=https://static.igem.org/mediawiki/2013/2/21/BYU2013-BiofilmAssayGraph1.jpg]]</div><br />
<br />
The above graph shows both the average pellet weight and the average absorbance readings for samples treated with 0, 2.5, 12.5, and 25 µL of α-Amylase. There is a distinct reduction in the amount of biofilm growth between untreated and treated samples, with samples treated with 25 µL α-Amylase showing a 65.8% decrease in biofilm formation after 48 hours. While this clearly shows the ability of α-Amylase to inhibit biofilm formation by ''V. cholerae'', further characterization is needed to determine the capacity of α-Amylase to degrade preexisting biofilms.<br />
<br />
</font><br />
<br><br />
<br />
<br><br />
<br />
<br></div>Perry721http://2013.igem.org/Team:BYU_Provo/Results/ExperimentalTeam:BYU Provo/Results/Experimental2013-09-28T02:21:10Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''Experimental Results'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Results|Results Overview]]<br />
<br />
: [[Team:BYU_Provo/Results/Judge|Judging Criteria]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Bronze|Bronze]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Silver|Silver]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Gold|Gold]]<br />
<br />
: [[Team:BYU_Provo/Results/Experimental|Experimental Results]]<br />
<br />
: [[Team:BYU_Provo/Results/Modeling|Modeling Results]]<br />
<br />
: [[Team:BYU_Provo/Parts|Parts Submitted]]<br />
<br />
</font><br />
<br />
| style="width: 81%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Phage Team==<br />
<br />
<br />
===Isolation of Phage Library===<br />
[[File:Changethecapsidsize.png|600px|center|link=https://static.igem.org/mediawiki/2013/3/36/Changethecapsidsize.png]]<br />
<br><br />
<br />
===Results of Mutant T4 Phage Isolation=== <br />
<br />
<br />
[[File:T4capsidvariation.png|600px|center|link=https://static.igem.org/mediawiki/2013/4/4e/T4capsidvariation.png]]<br />
<br />
<br />
[[File:T4CsClTube.png|600px|center|link=https://static.igem.org/mediawiki/2013/9/98/T4CsClTube.png]]<br />
<br><br />
<br />
===Results of Mutant T7 Phage Isolation===<br />
<br />
<br />
[[File:T7protocol.png|600px|center|]]<br />
<br />
As part of creating our bacteriophage library, we wanted to isolate both smaller and larger T7 bacteriophage. To do so, we first mutagenized T7 by growing it in the presence of 5-bromodeoxyuridine, a mutagenic base analog. This produces many mutant T7 bacteriophages, but they are hard to find amongst all the wild type T7. Therefore the phage purification team ran the mutagenized bacteriophage through a cesium chloride gradient. This separates the phage according to size, with the biggest traveling farther down. After determining where the mutant phage is in the gradient, we then plated the phage and looked for plaques that were smaller or larger than normal. Out of the 31 plaques we selected, two of them reproduced their plaque sizes, revealing that they were indeed mutant T7 bacteriophage! This can be seen in the two pictures below. The S4 plate with small phage produced larger plaques, while the L8 plate with large phage produced smaller plaques. The next step will be to get pictures of the phage with an electron microscope and sequence the genome to determine where the mutations occurred.<br />
<br />
In the two pictures below<br />
<br />
[[File:SmallResults.png|600px|center|]]<br />
<br />
[[File:LargeResults.png|600px|center|]]<br />
<br><br />
<br />
<br><br />
<br />
==Cholera Team==<br />
<br />
===Cholera Induces Bacteriophage Lambda From Lysogeny to its Lytic Cycle===<br />
<br />
[[File:byuk.jpg|250px|center|link=]]<br />
<br />
<br><br />
<br />
===Designing SdiA to be Specific for Cholera===<br />
<br />
[[File:BYUK Apr10-4.JPG|250px|center|link=]]<br />
<br />
In the beginning our plan was to transform into E.coli the most essential parts of the V.cholerae quorum sensing system. This was done, but our response proteins GFP and RFP did not work as expected.Shown above are colonies that were not supposed to be glowing. So we decided to try mutating the quorum sensing system E.coli already had to be able to only sense CAI-1 which is an auto-inducer specific to V.cholerae. <br />
<br />
[[File:BYU slide 4 cqss corrected.jpg|600px|center|link=]]<br />
[[File:BYU CqsS SdiA binding pocket.png|600px|center|link=]]<br />
<br><br />
<br />
===Biofilm inhibition by Amylase===<br />
<br />
The enzymatic activity of α-Amylase was characterized to determine its capacity to inhibit biofilm formation by ''V. cholerae''. Samples were prepared by adding 50 µL of ''V. cholerae'' culture to 1 mL of a high salt LB. The samples were then treated by the addition of purified α-Amylase in various concentrations and allowed to incubate at 30°C for 48 hours. After 48 hours the samples were examined and a distinct difference was seen in the amount of biofilm formed in the treated and untreated samples.<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssay1.jpg|450 px|link=https://static.igem.org/mediawiki/2013/2/2e/BYU2013-BiofilmAssay1.jpg]] [[File:BYU2013-BiofilmAssay2.JPG|249 px|link=https://static.igem.org/mediawiki/2013/e/ea/BYU2013-BiofilmAssay2.JPG]]</div><br />
<br />
<br />
The samples were then transferred to eppendorf tubes and centrifuged at 16,000 × g for two minutes, the supernatant was discarded to remove the growth media, and the samples were resuspended in 200 µL ddH2O. The samples were then stained with 50 µL of a 0.03% CV solution and allowed to incubate for five minutes. The samples were again centrifuged at 16,000 × g for two minutes and the supernatant containing excess CV was discarded. The pelleted biofilm was then washed with 800 µL of 95% EtOH without resuspension, centrifuged for 30 seconds, and the EtOH was discarded. The EtOH wash was repeated twice more for a total of three washes. The samples were then resuspended in 200 µL EtOH, transferred to a 96-well plate, and incubated for five minutes. The plate was then shaken for ten seconds and absorbance readings were taken for each sample at 540 nm, the absorbance wavelength of CV.<br />
<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssayGraph1.jpg|link=https://static.igem.org/mediawiki/2013/2/21/BYU2013-BiofilmAssayGraph1.jpg]]</div><br />
<br />
The above graph shows both the average pellet weight and the average absorbance readings for samples treated with 0, 2.5, 12.5, and 25 µL of α-Amylase. There is a distinct reduction in the amount of biofilm growth between untreated and treated samples, with samples treated with 25 µL α-Amylase showing a 65.8% decrease in biofilm formation after 48 hours. While this clearly shows the ability of α-Amylase to inhibit biofilm formation by ''V. cholerae'', further characterization is needed to determine the capacity of α-Amylase to degrade preexisting biofilms.<br />
<br />
</font><br />
<br><br />
<br />
<br><br />
<br />
<br></div>Perry721http://2013.igem.org/Team:BYU_Provo/Results/ExperimentalTeam:BYU Provo/Results/Experimental2013-09-28T02:18:04Z<p>Perry721: </p>
<hr />
<div>{{TeamBYUProvo}}<br />
<br />
<br><br />
<br />
{| width="100%"<br />
| colspan="2" | <font color="#333399" size="6" font face="Calibri"><br />
<br />
: '''Experimental Results'''</font><br />
<br />
<br><br />
<br />
|- valign="top"<br />
| style="width: 22%; background-color: transparent;"|<br />
<br />
<font color="#333399" size="3" font face="Calibri"><br />
<br />
: [[Team:BYU_Provo/Results|Results Overview]]<br />
<br />
: [[Team:BYU_Provo/Results/Judge|Judging Criteria]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Bronze|Bronze]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Silver|Silver]]<br />
<br />
: * [[Team:BYU Provo/Results/Judge#Gold|Gold]]<br />
<br />
: [[Team:BYU_Provo/Results/Experimental|Experimental Results]]<br />
<br />
: [[Team:BYU_Provo/Results/Modeling|Modeling Results]]<br />
<br />
: [[Team:BYU_Provo/Parts|Parts Submitted]]<br />
<br />
</font><br />
<br />
| style="width: 81%; background-color: transparent;"|<br />
<br />
<font face="Calibri" size="3"><br />
__NOTOC__==Phage Team==<br />
<br />
<br />
===Isolation of Phage Library===<br />
[[File:Changethecapsidsize.png|600px|center|link=https://static.igem.org/mediawiki/2013/3/36/Changethecapsidsize.png]]<br />
<br><br />
<br />
===Results of Mutant T4 Phage Isolation=== <br />
<br />
<br />
[[File:T4capsidvariation.png|600px|center|link=https://static.igem.org/mediawiki/2013/4/4e/T4capsidvariation.png]]<br />
<br />
<br />
[[File:T4CsClTube.png|600px|center|link=https://static.igem.org/mediawiki/2013/9/98/T4CsClTube.png]]<br />
<br><br />
<br />
===Results of Mutant T7 Phage Isolation===<br />
<br />
[[File:T7protocol.png|600px|center|]]<br />
<br />
<br />
As part of creating our bacteriophage library, we wanted to isolate both smaller and larger T7 bacteriophage. To do so, we first mutagenized T7 by growing it in the presence of 5-bromodeoxyuridine, a mutagenic base analog. This produces many mutant T7 bacteriophages, but they are hard to find amongst all the wild type T7. Therefore the phage purification team ran the mutagenized bacteriophage through a cesium chloride gradient. This separates the phage according to size, with the biggest traveling farther down. After determining where the mutant phage is in the gradient, we then plated the phage and looked for plaques that were smaller or larger than normal. Out of the 31 plaques we selected, two of them reproduced their plaque sizes, revealing that they were indeed mutant T7 bacteriophage! The next step will be to get pictures of the phage with an electron microscope and sequence the genome to determine where the mutations occurred.<br />
<br />
[[File:SmallResults.png|600px|center|]]<br />
<br />
[[File:LargeResults.png|600px|center|]]<br />
<br><br />
<br />
<br><br />
<br />
==Cholera Team==<br />
<br />
===Cholera Induces Bacteriophage Lambda From Lysogeny to its Lytic Cycle===<br />
<br />
[[File:byuk.jpg|250px|center|link=]]<br />
<br />
<br><br />
<br />
===Designing SdiA to be Specific for Cholera===<br />
<br />
[[File:BYUK Apr10-4.JPG|250px|center|link=]]<br />
<br />
In the beginning our plan was to transform into E.coli the most essential parts of the V.cholerae quorum sensing system. This was done, but our response proteins GFP and RFP did not work as expected.Shown above are colonies that were not supposed to be glowing. So we decided to try mutating the quorum sensing system E.coli already had to be able to only sense CAI-1 which is an auto-inducer specific to V.cholerae. <br />
<br />
[[File:BYU slide 4 cqss corrected.jpg|600px|center|link=]]<br />
[[File:BYU CqsS SdiA binding pocket.png|600px|center|link=]]<br />
<br><br />
<br />
===Biofilm inhibition by Amylase===<br />
<br />
The enzymatic activity of α-Amylase was characterized to determine its capacity to inhibit biofilm formation by ''V. cholerae''. Samples were prepared by adding 50 µL of ''V. cholerae'' culture to 1 mL of a high salt LB. The samples were then treated by the addition of purified α-Amylase in various concentrations and allowed to incubate at 30°C for 48 hours. After 48 hours the samples were examined and a distinct difference was seen in the amount of biofilm formed in the treated and untreated samples.<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssay1.jpg|450 px|link=https://static.igem.org/mediawiki/2013/2/2e/BYU2013-BiofilmAssay1.jpg]] [[File:BYU2013-BiofilmAssay2.JPG|249 px|link=https://static.igem.org/mediawiki/2013/e/ea/BYU2013-BiofilmAssay2.JPG]]</div><br />
<br />
<br />
The samples were then transferred to eppendorf tubes and centrifuged at 16,000 × g for two minutes, the supernatant was discarded to remove the growth media, and the samples were resuspended in 200 µL ddH2O. The samples were then stained with 50 µL of a 0.03% CV solution and allowed to incubate for five minutes. The samples were again centrifuged at 16,000 × g for two minutes and the supernatant containing excess CV was discarded. The pelleted biofilm was then washed with 800 µL of 95% EtOH without resuspension, centrifuged for 30 seconds, and the EtOH was discarded. The EtOH wash was repeated twice more for a total of three washes. The samples were then resuspended in 200 µL EtOH, transferred to a 96-well plate, and incubated for five minutes. The plate was then shaken for ten seconds and absorbance readings were taken for each sample at 540 nm, the absorbance wavelength of CV.<br />
<br />
<br />
<div style="text-align:center;">[[Image:BYU2013-BiofilmAssayGraph1.jpg|link=https://static.igem.org/mediawiki/2013/2/21/BYU2013-BiofilmAssayGraph1.jpg]]</div><br />
<br />
The above graph shows both the average pellet weight and the average absorbance readings for samples treated with 0, 2.5, 12.5, and 25 µL of α-Amylase. There is a distinct reduction in the amount of biofilm growth between untreated and treated samples, with samples treated with 25 µL α-Amylase showing a 65.8% decrease in biofilm formation after 48 hours. While this clearly shows the ability of α-Amylase to inhibit biofilm formation by ''V. cholerae'', further characterization is needed to determine the capacity of α-Amylase to degrade preexisting biofilms.<br />
<br />
</font><br />
<br><br />
<br />
<br><br />
<br />
<br></div>Perry721