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

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Week 7: June 10 - June 14

This week involved transforming the part BBa_K648013 received by request from the Registry of Standard Biological Parts. This part is a GFP reporter that contains the RFC 25 standard prefix and suffix. This prefix and suffix will allow us to create a fusion protein with GFP and the coils to be used in our testing purposes. After successful transformation of the part the plasmid was isolated via miniprep and its presence was confirmed through a restriction digest. The part was also sequence confirmed to indeed verify that there were no unexpected changes to the nucleotide sequence.

Week 8: June 17 - June 21

Research efforts were devoted to the Ferritin system during this week. Flooding in the city of Calgary interrupted the planned lab work for the beta-lactamase and coil group.

Week 9: June 24 - June 28

In order to use beta-lactamase as a potential reporter system it was necessary to use designed primers to PCR out the gene from the backbone of the pSB1A3 plasmid (it is present here as it conveys ampicillin resistance). Three primers were designed in order to retrieve the beta-lactamase gene just by itself as well as having it with a His-tag in order to aid with protein purification. After the PCR was performed the resulting DNA was run on a gel to confirm the gene size (Figure 2). This PCR appeared to be successful based on size (900-bp and 927-bp for the beta-lactamase and beta-lactamase with His-tag respectively) but after sequencing it was determined one of the primers contained a design error resulting in a truncated gene. The primers will be redesigned in order to successfully achieve the extraction of the beta-lactamase gene from the pSB1A3 plasmid.

The first BioBrick construction was attempted this week as well. The goal was to put the RFC25 fusion standard GFP (BBa_K648013) behind the IPTG inducible LacI promoter (with a ribosome binding site) (BBa_J04500). This construction however did not yield any colonies after ligation and transformation were performed. It is recommended the next time this construction be performed that the amount of vector DNA added to the ligation reaction be increased from 5 µL to 7 µL to correct for the dilution of the vector DNA during the Antarctic phosphatase treatment performed. BBa_J04500 in pSB1C3 was transformed this week so that we could have this part available with a chloramphenicol resistant plasmid. The colony PCR for this part was successful (Figure 3) ergo overnight cultures were prepared on the weekend so that a miniprep of the plasmid could be performed the next week.

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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