Linker Journal

Week 1: May 1 - May 3

This week we attended the appropriate safety courses required by our University to work in the lab. Besides that, the undergraduate supervisors presented about the principles of Genetics and Synthetic Biology to the new team members.

Week 2: May 6 - May 10

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

Week 3: May 13 - May 17

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

Week 4: May 20 - May 24

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.

IAAL E3/K3 Coil Helical Wheel Diagram

Figure 1. A helical wheel representation of the IAAL E3/K3 coiled-coil heterodimer viewed as a cross-section based off of a similar figure created by Litowski and Hodges (2002). The peptide chain propagates into the page from the N to the C terminus. Hydrophobic interactions between the coils are indicated by the clear wide arrows. The intermolecular electrostatic interactions between the coils are displayed by the thin curved arrow (eg. Between Glu15 and Lys20)Letters a, b, c,and d designate the positions of IAAL repeat in the heptapeptide. THe e and g positions are occupied by the charged residues.

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 5: May 27 - May 31

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 6: June 3 - June 7

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 test the 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 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 β-lactamase and coil group.

Week 9: June 24 - June 28

The first BioBrick construction was attempted this week. 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 2) ergo overnight cultures were prepared on the weekend so that a miniprep of the plasmid could be performed the next week.

J04500 Confirmation PCR

Figure 2. A colony PCR of potential colonies containing the part J04500. Standard BioBrick primers were used. Bands between 400 and 500-bp indicate the successful amplification of these parts. +C indicates a positive control making use of the RFP coding device J04450 which has an expected size of about 1300-bp when using BioBrick primers. Some non-specific amplification is seen within this control. The gel was 1% agarose and was run at 100 V for an hour.

Week 10: July 1 - July 5

The overnight cultures produced on the weekend were successfully miniprepped producing DNA stocks of the LacI promoter BBa_J04500 (in pSB1C3). Restriction digests of this part confirmed a successful transformation and miniprep. An initial attempt was made to make a construct composed of the LacI promoter BBa_J04500 followed by the RFC25 fusion standard GFP (BBa_K648013). This construction produced few viable colonies. One colony appeared to have the expected band size after colony PCR (Figure 3) however this result could not be replicated. This construction will be attempted again in the future.

J04500 and K648013 Ligation Confirmation PCR

Figure 3. A colony PCR of potential colonies containing the part J04500 + K648013. Standard BioBrick primers were used. The band about 1100-bp in size indicates the successful amplification of this part. +C indicates a positive control making use of the RFP coding device J04450 which has an expected size of about 1300-bp when using BioBrick primers. No contamination is observed in the NTC (no template control). The gel was 1% agarose and was run at 100 V for an hour.

Also received this week were the synthesized coil genes ordered from BioBasic. These genes are the K coil, E coil, K coil with a His-tag, and E coil with a His-tag. These genes were successfully transformed into our E. coli cells and were miniprepped successfully. These genes are in the pUC57 (pUC57 PDF Information Sheet) vector and will have to be plasmid switched into pSB1C3 for submission to the registry.

Week 11: July 8 - July 12

Confirmation digests were performed upon the coil parts miniprepped last week in order to confirm the presence of the coils (Figures 5). These coils can be seen as the faint bands at approximately 100-bp in size on the gel. Digestions like these could be performed as the synthesized genes contain the RFC 25 prefix and suffix. These digestions revealed that the coil genes are indeed present in the minipreps produced. Glycerol stocks were made of our transformed cells for organized long term storage.

Coil Confirmation Digest

Figure 4. Confirmation digests of the K coil, E coil with His-tag and the E coil parts within the pUC57 vector using the NotI enzyme. Bands are all expected to be approximately 100-bp and are indicated by the orange arrows due to their faintness. The –C lane is a negative control. +C indicates a positive control making use of the RFP coding device J04450 which has an expected size of about 1100-bp when digested. The gel was a 2% agarose gel and was run at 80 volts for 90 minutes.

A construction attempted this week consited of placing the RFC25 fusion standard GFP (BBa_K648013) gene behind the promoter BBa_J04500. Also attempted were plasmid switches of all of the coil genes ordered. Colonies were seen for all of the transformed ligation mixtures for these constructions. Colony PCR of these transformed colonies only produced potentially viable results for the J04500 + K648013 construct. The K coil, K coil His-tag and E coil His-tag plasmid switches also displayed bands around the expected size during colony PCR.

Week 12: July 15 - July 19

This week contained some disappointment as the BioBrick constructions continued to have limited efficacy. The confirmation digests of the construct that showed some promise during last week’s colony PCR (J04500 + K648013) and the plasmid switches (K coil, K coil His-tag and E coil His-tag) did not produce expected results. One rather notable issue is the ~200-bp size difference seen in the J04500 + K648013 from the expected size of 955-bp. The difference appears to suggest that the LacI promoter is not present. This hypothesis was confirmed with sequencing results. This would suggest that the wrong enzymes were used during construction or that there is potential contamination of the enzyme buffers used.

A replicate of these constructions this week also produced similar results. Due to this it is recommended that new buffers be used for further constructions. Some of the difficulty present with plasmid switching the coils is due to their small size however. Since the coils themselves are at minimum approximately 60-bp in size. This is not kinetically favorable for BioBrick construction. It may be wise to primer on the coils to appropriate constructs when the time comes for their addition. Also present in our constructions are re-ligations of the original vector. Due to this the time for phosphatase treatment will be increased from two to three hours to limit this issue.

Week 13: July 22 - July 26

Proceeding with the recommendations from last week regarding BioBrick constructions the plasmid switches of the coil genes into pSB1C3 were attempted again. Based off of the colony PCR there were some potential completed parts for the plasmid switches. Verification digests of the miniprepped plasmids from these colonies combined with sequencing revealed the successful plasmid switch of the E coil with a His-tag (Figure 5).

E coil with His-tag Plasmid Switch

Figure 5. Confirmation digests of the K coil, E coil with His-tag, K coil with His-tag and the E coil parts within the pUC57 vector using the NotI enzyme are present in the respectively labelled lanes. These digests were used to confirm if older coil stocks were suitable for construction. Also present are the confirmation digests of the K coil, E coil with His-tag, and K coil with His-tag after an attempted plasmid switch into pSB1C3. The expected bands were observed for the plasmid switch of the E coil with the His-tag. Due to the faintness of the bands they are indicated with orange arrows (reference stock plasmid bands are also indicated). Positive and negative control lanes are present on another gel. The gel was a 2% agarose gel and was run at 80 volts for 90 minutes.

Week 14: July 29 - August 2

We attempted plasmid switching the coils into pSB1C3 however our attempt was unsuccessful. None of the plates had any colonies. This was attempted twice.

Week 15: August 5 - August 9

A test to see whether our ligase was functional was conducted in order to trouble shoot the problems with construction we were facing. Our ligase works.

Week 16: August 12 - August 16

No work on linkers. Work on reporter and protein work was attempted for our constructs.

Week 17: August 19 - August 23

No work on linkers. Work on reporter and protein work was attempted for our constructs

Week 18: August 26 - August 30

Unsuccessful plasmid switches of the coils.

Week 19: September 2 - September 6

Unsuccessful plasmid switches of the coils.

Week 20: September 9 - September 13

We were finally able to switch the coils into pSB1C3. We were able to successfully confirm the results of this plasmid switch using PCR confirmation, confirmation digestion (Figure 6), and sequencing.

Figure 6. Confirmation digests of the E-coil, K-coil and K-coil with His-tag into pSB1C3. -C indicates a negative control. +C indicates a positive control making use of the RFP coding device J04450 which has an expected size of about 1100-bp when digested. The gel had a 3% agarose content and was run at 90 volts for 90 minutes.

Week 21: September 16 - September 20

DNA submission of the coils after we got sequence verification. We are also working on using the coils in our system to test whether they self assemble.

Week 22: September 23 - September 27

Wiki Freeze!

Week 23: September 30 - October 4

Practice, practice, practice for our Regional Jamboree presentation!

Week 24: October 7 - October 11

We performed a co-immunoprecipitation to prove that our coils actually bind to each other. Figure 7 shows our results. The immunoprecipitation worked! Therefore our coils bind to each other and can be used in our system!

Figure 7. Immunoprecipitation assay showing that the E and K coils interact with each other. The first three lanes show E coil, K coil and E coil with K coil pulled down with IgG (control) antibody. The last three lanes were pulled down with a anti-GFP antibody and probed with a anti-his antibody. The E coil is fused to GFP and the K coil is fused to TALE with a his tag. Therefore, if we pull down with GFP and probe with his-antibody a band would appear the size of TALE. This indicates that both the GFP and the TALE are present and that is possible upon coil-coil interaction.

Week 25: October 14 - October 18

Organized what parts will need to be submitted. Planned experiments to test our whole system.

Week 26: October 21 - October 25

We experimented to determine whether the linkers are effective in our FerriTALE system, compared to direct fusion. Figure 8 shows that the use of the E and K coils allow our system to work optimally. As predicted by our modelling,direct fusion is not desirable, meaning the ferritin and TALEs need to be linked.

Recombinant Prussian Blue FerritinMole Balanced

Figure 8. Samples of our parts that were converted to Prussian Blue ferritin were mole balanced in order to ensure that the same number of effective ferritin cores are present in every sample. Additionally the ferritin-coil fusion was incubated with the TALE-coil fusion part in order to allow their binding for a separate trial. Negative controls include unconverted recombinant ferritin, bovine serum albumin and a substrate only control. Samples were incubated with a TMB substrate solution for 10 minutes at a pH of 5.6. Absorbance readings were taken at the 10 minute time-point at a wavelength of 650 nm. An ANOVA (analysis of variants) was performed upon the values to determine that there was statistical difference in the data gathered (based off of three replicates). A t-test was then performed which determined that the * columns are significantly different from the ** column (p=0.0012). Neither * column is significantly different from each other (p=0.67).

Week 27: October 28 - November 1

It's our favourite time of the year; Wiki Freeze! See you in Boston.