Linker Journal

Week 1: May 1 - May 3

This week we participated in a general molecular biology workshop to refresh our memory of techniques used in molecular biology.

Week 2: May 6 - May 10

We continued the molecular biology workshop. This week we also divided up into our respective groups for the project and decided research priorities.

Week 3: May 13 - May 17

This week we started to perform literature searches for our element of the project. Our goal is to find a way to link the TALE and ferritin elements of the project. We have decided that using a two part system is optimal as this would allow the ferritin subunits to self-assemble without having a TALE protein attached that could potentially interfere with this process. In order to accomplish this we have decided to use coiled-coils. The synthetic IAAL E3 and IAAL K3 coils (Litowski and Hodges, 2002) have been selected to accomplish this. These coils make use of hydrophobic regions composed of leucine and isoleucine to bind to each other (Figure 1). The specificity of these coils is conveyed by the presence of glutamic acid and lysine residue that limit the binding to the formation of coil heterodimers. Based off of results from previous ferritin fusions (Kim et al., 2011) it was decided that a linker sequence was needed between the coils and their respective proteins to prevent any steric hindrance due to the size of the ferritin and TALE proteins. A flexible glycine rich linker lacking protease cut sites was selected from the registry (BBa_K157013)

Week 4: May 20 - May 24

We continued to perform literature searches this week for our project. This week our main focus was on determining ways we could characterize our coils and finding additional useful properties of coils. Many papers make use of circular dichroism in order to detect if the coils are able to bind to each other. This technique requires the use of a spectropolarimeter and a fair amount of knowledge to complete so it may not be the most ideal technique in our situation given the timelines we are working on. Another option consists of using FRET (Förster resonance energy transfer) to measure the binding of the coils. The idea is to have an individual fluorophore attached to each coil so that when the coils are bound the process of FRET can occur and light emission will be observed from the fluorescent protein that is having energy being transferred to it from the other fluorescent protein (Apostolovic and Klok, 2008). This technique can be difficult to perform however so it will likely be maintained as a backup characterization technique. One interesting properties of coils is that they are sensitive to acidic pH levels (Apostolovic and Klok, 2008). This warrants examination once we have our coils complete and purified as a protein as this pH sensitivity could be problematic for our system or it may act as a benefit.

Week 5: May 27 - May 31

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Week 6: June 3 - June 7

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Week 7: June 10 - June 14

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Week 8: June 17 - June 21

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Week 9: June 24 - June 28

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Week 10: July 1 - July 5

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Week 11: July 8 - July 12

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Week 12: July 15 - July 19

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Week 13: July 22 - July 26

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Week 14: July 29 - August 2

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Week 15: August 5 - August 9

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Week 16: August 12 - August 16

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Week 17: August 19 - August 23

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Week 18: August 26 - August 30

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Week 19: September 2 - September 6

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Week 20: September 9 - September 13

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Week 21: September 16 - September 20

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Week 22: September 23 - September 27

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Week 23: September 30 - October 4

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