Team:CSU Fort Collins/Notebook/HVP/Jun

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   <center><h1>Breakdown Daily Notes</h1>
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   <center><h1>High-Value Product Daily Notes</h1>
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     <h2>JULY</h2>
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     <h2>JUNE</h2>
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    <h4>Tuesday, July 1 - Wednesday, July 16</h4>
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    Researched which promoters, primers, sequences, and assembly techniques to use and developed a plan of action.
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     <h4>Thursday, July 17</h4>
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     <h4>Monday, June 2 - Friday, June 6</h4>
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     Designed primers to connect biobrick lab promoter and the FadD gene from <u>E. coli</u>.
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     Researched potential products and decided on the addition of the mevalonate pathway in order to direct <u>E. coli</u> to terpenoid production.
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     <h4>Friday, July 18</h4>
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     <h4>Monday, June 9 - Monday, June 16</h4>
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     With competent cells, used the transformation protocol to add our two different lac promoters to <u>E. coli</u> and grew overnight at 37 &#176;C. Used lac promoters from the biobrick distribution kit (2013 iGEM Distribution Kit, Plate 5, Well 1D (ampicillin resistance) and 2014 iGEM Distribution Kit, Plate 3, Well 4G (chloramphenicol resistance)).
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     Researched the mevalonate pathway in E. coli and sequenced the genes to be inserted into puc19. Designed primers as specified in the <a href='/Team:CSU_Fort_Collins/Notebook/Protocols=Gibson'>Gibson Assembly Protocol</a> for transformation of the mevalonate pathway from yeast into <u>E. coli</u>.  
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     <h4>Sunday, July 19</h4>
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     <h4>Thursday, June 19</h4>
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     Removed plates from the incubator and stored at 4 &#176;C.
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     Ran a <a href='Team:CSU_Fort_Collins/Notebook/Protocol/Gel'>gel electrophoresis</a> to try and confirm presence of DNA in obtained yeast samples. No results.  
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     <h4>Monday, July 21</h4>
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     <h4>Tuesday, June 24</h4>
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     The plate using the promoter from 1D had no growth. The plate with the promoter from 4G had two small colonies. We replated the colonies using the rest of our transformed competent cells. Incubated again overnight at 37 &#176;C.
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     Mixed and autoclaved two 500 mL bottles of LB Broth with Agar and ampicillin. Poured two sleeves of plates to use for culturing.
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     <h4>Tuesday, July 22</h4>
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     <h4>Wednesday, June 25</h4>
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     The plate using the promoter from 1D still had no growth. The plate with the promoter from 4G had a lawn. Streaked the 4G plate to create individual colonies. Incubated overnight at 37 &#176;C.
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     Took a small sample of E. coli with puc19 from a previous colony and suspended in LB Broth. Shook at 225 rpm in incubator at 37&#176; C overnight to culture.
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     <h4>Wednesday, July 23</h4>
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     <h4>Thursday, June 26</h4>
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     The 4G plate had good growth with many individual colonies. We created two 2 mL overnight cultures using 1 mL LB/1 &#956;L CM and a colony from our streaked plate. Incubated overnight at 37 &#176;C and 225 rpm.
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     Completed a miniprep of E. coli with puc19 from the overnight culture. Followed <a href='/Team:CSU_Fort_Collins/Notebook/Protocol/Miniprep'>plasmid miniprep protocol</a> as given in the Informatics (Life Technologies) kit.
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    <h4>Thursday, July 24</h4>
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     <h4>Friday, June 27</h4>
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    Removed the tubes from the incubator and stored at 4 &#176;C.
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     Completed PCR with the 5 pieces according to the mixture of New England Biotechnologies' Q5 2X Master Mix protocol. Ran a gel electrophoresis to confirm success of PCR. Amplification of PMK unsuccessful. Amplification of all other portions of the plasmid successful.
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     <h4>Friday, July 25</h4>
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     The primers for FadD gene came in. Created a 100 &#956;M stock solution. Diluted by 10X to make a working solution. Following the PCR protocol outlined in the Cloning a Gene into a Plasmid protocol, PCRed 4 replicates. Used a 68 &#176;C annealing temperature and a 1 minute 30 seconds extension. Stored the PCR product tubes at 4 &#176;C.
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    Miniprepped 1 mL of overnight cultures to extract the DNA containing the desired lac promoter as directed in the <a href='/Team:CSU_Fort_Collins/Notebook/Protocols=Miniprep'>Miniprep Protocol</a>. The products of the miniprep were stored at -20 &#176;C.
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    Created 2 more overnight cultures using the same protocol as before, this time seeding them using what was left of the previous overnight cultures.
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    <h4>Tuesday, July 29</h4>
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Used two <a href='/Team:CSU_Fort_Collins/Notebook/Protocols=Gel'>gel electrophoresis</a> protocols to determine if the PCR worked. Both gels failed.
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<h4>Wednesday, July 30</h4>
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Reran PCR using a different temperature for the extension step. Ran another gel to determine success of PCR. This gel also failed.
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<h4>Thursday, July 31</h4>
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Reran PCR using previously isolated <u>E. coli</u> genome and various extension temperatures in a gradient. Ran another gel, this gel also failed.
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    <h4>Monday, June 30</h4>
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    Modified the PCR thermalcycler program (but used the same 50 &#956;L mixture) and ran a gel to check results. All attempts unsuccessful - suspected due to lack of presence of SYBR green in initial mixture.
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     <center><a href='/Team:CSU_Fort_Collins/Notebook/Biosensor/Aug' id='navi' style='margin-left:-20px; margin-right:40px'> Previous</a> <a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Aug' id='navi'>Next </a></center>
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Revision as of 19:01, 14 October 2014

High-Value Product Notes - June

High-Value Product Daily Notes

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Jun

pMBI Assembly

July

pMBI and pMevT Assembly

August

Troubleshoot Everything!

September

Plan B

JUNE

Monday, June 2 - Friday, June 6

Researched potential products and decided on the addition of the mevalonate pathway in order to direct E. coli to terpenoid production.

Monday, June 9 - Monday, June 16

Researched the mevalonate pathway in E. coli and sequenced the genes to be inserted into puc19. Designed primers as specified in the Gibson Assembly Protocol for transformation of the mevalonate pathway from yeast into E. coli.

Thursday, June 19

Ran a gel electrophoresis to try and confirm presence of DNA in obtained yeast samples. No results.

Tuesday, June 24

Mixed and autoclaved two 500 mL bottles of LB Broth with Agar and ampicillin. Poured two sleeves of plates to use for culturing.

Wednesday, June 25

Took a small sample of E. coli with puc19 from a previous colony and suspended in LB Broth. Shook at 225 rpm in incubator at 37° C overnight to culture.

Thursday, June 26

Completed a miniprep of E. coli with puc19 from the overnight culture. Followed plasmid miniprep protocol as given in the Informatics (Life Technologies) kit.

Friday, June 27

Completed PCR with the 5 pieces according to the mixture of New England Biotechnologies' Q5 2X Master Mix protocol. Ran a gel electrophoresis to confirm success of PCR. Amplification of PMK unsuccessful. Amplification of all other portions of the plasmid successful.

Monday, June 30

Modified the PCR thermalcycler program (but used the same 50 μL mixture) and ran a gel to check results. All attempts unsuccessful - suspected due to lack of presence of SYBR green in initial mixture.



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