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 transcription activator-like effector(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

Based on the research we have done in the previous weeks we have decided to plot out some experiments that will help us characterize our coils. We plan make use of a Ni-NTA column and His-tags on our coils to characterize our coils. The idea is that we can have a His-tag located on one of the coils and this coil will bind to the column via the His-tag. The second coil will be fused to protein such as GFP (BBa_K648013). This second coil will not have a His-tag and when passed through the column it ideally should bind to the other coil already immobilized via a His-tag. By varying the conditions within the column we should be able to characterize the binding of the coils. If the coil fused to GFP falls off of the other coil it should be located in the collected fluid. The presence of the GFP can be detected by both fluorescence and SDS-PAGE. Three proposed experiments that we have making use of this are detailed below:

• The first test will just test the binding of the coils to each other under standard coil binding conditions (pH = 7). A K-coil will be bound to a drip Ni-NTA column via a 6x His-tag C-terminus fusion. An E-coil with an N-terminus GFP fusion can then interact with the K-coil that is bound to the column. If the interaction is successful the GFP will not be found in high quantities in the wash steps. The GFP fusion should elute when imidazole is added to the column. The levels of GFP can be verified using both emission measurements and SDS-PAGE.
• The second test will make use of this column but will measure the effect of pH on the binding of the coils. Different columns will be set up with different pH washes (3, 5, 7 and 9). It has been previously reported that the coils begin to dissociate at a pH of 5 (Apostolovic and Klok, 2008).
• The third test will make use of a spin Ni-NTA column. This test will testthe ability of the heterodimer coiled-coils to resist shearing due to applied forces. The columns will undergo centrifugation at different steps after the proteins have been bound to determine if the forces present have any effect on the binding of the coils.

Week 6: June 3 - June 7

Enter Text Here

Week 7: June 10 - June 14

Enter Text Here

Week 8: June 17 - June 21

Enter Text Here

Week 9: June 24 - June 28

Enter Text Here

Week 10: July 1 - July 5

Enter Text Here

Week 11: July 8 - July 12

Enter Text Here

Week 12: July 15 - July 19

Enter Text Here

Week 13: July 22 - July 26

Enter Text Here

Week 14: July 29 - August 2

Enter Text Here

Week 15: August 5 - August 9

Enter Text Here

Week 16: August 12 - August 16

Enter Text Here

Week 17: August 19 - August 23

Enter Text Here

Week 18: August 26 - August 30

Enter Text Here

Week 19: September 2 - September 6

Enter Text Here

Week 20: September 9 - September 13

Enter Text Here

Week 21: September 16 - September 20

Enter Text Here

Week 22: September 23 - September 27

Enter Text Here

Week 23: September 30 - October 4

Enter Text Here