Team:CU-Boulder/Notebook

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Notebook

Week 1: 5/8/16-5/14/16

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For the first week, the team participated in “iGEM boot camp” in which we quickly went through the procedures and equipment that we would be using throughout the summer. This entailed the procedures for PCR amplification, gel electrophoresis, cloning, and DNA sequencing. We extracted the Eut SMNLK via an agar stab provided from the Schmidt- Dannert lab and PCR amplified the Eut S gene using the biobrick primers and cloned on the high, low, and lac promoters onto the 1C3 backbone prior to the ligation of the Eut S gene onto these modified backbones. The 1C3 backbone was taken from the 2016 iGEM registry. Finally, we ligated the Eut S- high, low, and lac onto PSB1C3, the bio-brick standard backbone.

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Week 2: 5/15/16-5/21/16

Gel.1 - Lane 2: eGFP-Hi 1c3 Lane 3: eGFP-Hi 1c3 Lane 4: ladder Lane 5: eGFP-Lac 1c3 Lane 6: eGFP-Lac 1c3

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Week 3: 5/22/16-5/28/16

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Week 4: 5/29/16-6/4/16

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Week 5: 6/5/16-6/11/16

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Week 6: 6/12/16-6/18/16

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Week 7: 6/19/16-6/25/16

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Week 8: 6/26/16-7/2/16

Gel.2 - Lane 2: bad cut with E Lane 3: Nep-Lo 1k3 Lane 4: Nep-Lo 1k3 Lane 5: Ladder Lane 6: eGFP-Hi 1k3 Lane 7: eGFP-Hi 1k3

BSuccessful microscopy has confirmed the viability of EutS and EutC-eGFP in E.Coli, but the laser used to excite eGFP may also cause conformational change of azo-benzene. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance

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Week 9: 7/3/16-7/9/16

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Week 10

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Week 11: 7/10/16-7/16/16

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Week 12: 7/17/16-7/23/16

Gel.3 - Lane 2: eGFP-Hi 4A5 Lane 3: eGFP-Hi 4A5 Lane 4: Ladder Lane 5: eGFP-Hi 4A5 Lane 6: eGFP-Hi 4A5. Lane 3 and Lane 5 look good

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Week 13: 7/24/16-7/30/16

Gel.4 - Lane 1: Ladder Lane 2: EutS- Hi 6A1 Lane 3: EutS-Hi 4A5 Lane 4: EutS-Hi 4A5 Lane 5: Ladder Lane 6: eGFP-Hi Lane 7: eGFP-Hi. eGFP is good

BBBBBBBBBBBBB BBBBB BBBBBBBBBBBBBBB BBBBBBBBBBBBBBBBB BBBBBBB BBBBB BBBBBBBB BBBBB bbbbbb bbb bbbbb bb bb bbbbbbbbbbb bb bbbbbbbb

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Week 14: 7/31/16-8/6/16

Gel.5 - Lane 1: Ladder Lane 2: EutS-Hi Lane 3: EutS-Hi Lane 4: EutS-Hi Lane 5: Ladder Lane 6: EutS-Lac 6A1 Lane 7: EutS-Lac 4A5 Lane 8: EutS-Lac 4A5

Successful microscopy has confirmed the viability of EutS and EutC-eGFP in E.Coli, but the laser used to excite eGFP may also cause conformational change of azo-benzene. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.

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Week 15: 8/7/16-8/13/16

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Week 16

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Week 17: 8/14/16-8/20/16

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Week 18

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Week 19

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Week 20

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Week 21

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Week 22

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Week 23

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Week 24

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Week 25

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Week 26

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Week 27

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