Engineering

Cycle 1

Design

In order to fuse the cell penetrating peptides (CPPs) to our ferritin, we designed primers that have an overlapping part complementary to the ferritin. The CPPs and the glycine-serine linker were included by a non-overlapping region for introducing them during the synthesis of the PCR (fig. 1).

Build

We ordered the primers and promptly started the mutagenesis following the protocol that we used for mutating the BsaI and SapI restriction sites beforehand. The annealing temperature of 62.2 °C was calculated using the Thermofisher Tm-calculator.

Test

Afterwards, we examined the outcome by applying the samples to an agarose gel. 6,000 bp bands were expected, however our first mutagenesis failed (fig. 2).

Learn

The protocol we used proved to be unsuitable for the implementation of the CPPs. We had to optimize the protocol.
We assumed that we may have used an incorrect annealing temperature, hence we had to find a way of testing that.

Cycle 2

Design

To save resources, we agreed to continue with TAT-CPP for the optimzation of our mutagenesis protocols.
To test the impact of the annealing temperature on our results, we decided to conduct a gradient PCR to see would if the results would validate or dismiss our assumption.

Build

We created a temperature gradient of 5 °C centered around 60 °C to broaden the range of annealing temperatures tested.

Test

We examined the second mutagenesis’ success by running the samples on an agarose gel and as before, we did not obtain the expected results of bands at 6,000 bp (fig. 3).

Learn

The results using a range of annealing temperatures did not deliver optimal results. We concluded that we would have to adjust another parameter for success.

Cycle 3

Design

Next, we altered the parameter concerning the duration of the synthesis step of our PCR program. Additionally, we include varying amounts of DNA to understand the impact of the templates’ concentration in the reaction.

Build

The guideline for the synthesis according to NEB was to plan 20-30 seconds per kb. Our plasmid is about 6,000 bp large, which is why we decided to adjust our synthesis duration to 3.5 minutes instead of 2 minutes. We began with samples of 10 ng of DNA and ended with 70 ng. Concerning the annealing temperature, we returned to the first one we used since it was calculated based on the annealing part of our primers.

Test

As before, we used an agarose gel to confirm the success of the mutagenesis and, this time, bands were visible at the expected height of 6,000 bp (fig.4.)!

Learn

The impact of the template concentration used in the reaction was minimal.
But we now learned that, having a larger plasmid, incorperating a longer synthesis duration is required for optimal results. For further experiments, we would amplify plasmids for a duration of 3.5 minutes each to be on the safe side.

Engineering success!

Now that we had optimized our method, we repeated the CPP-implementation of all three CPPs as in our first step. We adjusted the synthesis time, increasing it from 2 minutes to 3.5 minutes. Again, we checked the results using an agarose gel and were delighted to see the results (fig. 5)!

Then, it was possible for us to continue with the transformation of E. coli with the PCR product, followed by colony PCR and sequencing, and could then obtain the correctly mutated plasmid. This plasmid we could then use for expressing our CPP-ferritin constructs!

Cycle 1

Design

The protocol for the purification of ferritin contains three steps for the purification: First heat precipitation, then ion exchange chromatography (IEC), followed by size exclusion chromatography (SEC). We used the protocol that Prof. Dr. Tobias Beck provided to us.

Build

For the ion exchange chromatography, following the protocol provided by Beck, we used an anion exchange column. The proteins were dissolved in the lysis buffer used for the heat precipitation with a pH value of 7.5. We let two samples of each of the four constructs - TAT-Ferritin (BBa_K469005), R9-Ferritin (BBa_K4669004), R12-Ferritin (BBa_K4669006), and WT-Ferritin (BBa_K4669000) - run over the column.

Test

We saw peaks in the chromatograms for all samples (fig. 6-9), one sample of each construct is displayed. These peaks corresponded to our expectations with an elution at around 35 mS/cm. We continued with the fractions of these peaks, concentrated them and applied them on an SDS-PAGE. Unfortunately, clear bands were only visible for WT-Ferritin (fig. 10). Our TAT-, R9-, and R12-Ferritin constructs were evidently lost during IEC purification.

Learn

We discussed our method again and quickly found an error source to address. When purifying our TAT-, R9- and R12-Ferritin samples, we did not consider the need to adjust the pH of our lysis and elution buffer. This is due to the fact that the attached CPPs would change the isoelectric point (pI) of our protein (tab. 1). Therefore our constructs might not have been as negatively charged as we assumed, leading to an earlier elution.

Tab. 1: The isoelectric points of WT-Ferritin and all CPP-Ferritin.

Construct	Isoelectric point
WT-Ferritin	5.3
TAT-Ferritin	6.87
R9-Ferritin	6.59
R12-Ferritin	7.91

For changing pIs, the pH of the buffer that the proteins are dissolved in has to be adapted in order to keep them negatively charged and to have a well functioning anion exchange.

Cycle 2

Design

For the optimization of the IEC purification step we adapted the lysis buffer by changing its pH value. Since we had prepared a number of heat precipitated samples, we decided on repeating this step for R9- and TAT-Ferritin. With R12-Ferritin’s pI being the highest, we concluded that we would exclude R12-Ferritin, until our method was successfully optimized.

Build

For both R9- and TAT-Ferritin we now used a buffer with a pH value of 9.

Test

Unfortunately, despite that the protein concentrations of the samples were similar to the samples used before, we could not detect any peak (fig. 11).

Learn

Somehow, we lost our protein again. Currently, we are still in the process of finding new approaches for optimizing the protocol for protein purification. Due to the time constraints, we were only able to purify WT-Ferritin and have not been successful in finding our lost protein yet.

Cycle 1

Design

For expressing the ferritin with a non-canonical amino acid, we had to introduce the Amber-codon into its backbone. Besides adding the non-canonical amino acid to the medium, we had to transform the expression strain with a second plasmid: the helper plasmid containing the aminoacyl-tRNA synthetase and the corrosponding tRNA. Both are essential when expressing ferritin.
We designed a plasmid with a chloramphenicol resistance, containing an origin of replication (ORI) that differs from the one on the WT-Amber-Ferritin (BBa_K4669007) and the TAT-Amber-Ferritin plasmids (BBa_K4669008) (fig. 12-14).

The backbone was available to us in the lab. We only had to add the BsaI restriction recognition sites to the end of it by PCR. The insert was divided into three fragments that we ordered. These were designed for a Golden Gate Assembly (GGA) (fig. 15).

Build

We then started our first attempt of the GGA reaction, using the following protocol:

Reagents	Volume
p15A backbone (40 ng/µL)	1.64 µL
Fragment 1 (50 ng/µL)	2.19 µL
Fragment 2 (50 ng/µL)	2.10 µL
Fragment 3 (25 ng/µL)	1.13 µL
T4 DNA ligase buffer (10X)	2 µL
NEB GGA Mix BsaI	1 µL
MiliQ water	To 20 µL
Total	20 µL

Temperature	Time
37 °C	10 min
16 °C	10 min
Go to 1, repeat 9x
37 °C	20 min
60 °C	20 min
4 °C	hold

We then transformed the products into DH5α cells, followed by a colony PCR for evaluation of the colonies.

Test

PCR fragments of about 900 bp were expected. In our experience, colony PCR are fairly pronounced. However, our obtained fragments were rather slim (fig. 16). But, since we had clear products at the expected height, we sent the plasmids of GGA #2-5 to sequencing. The results came back negative.

Learn

When repeating the program, we obtained negative results. We decided to optimize the program.

Cycle 2

Design

We used the same volumes, but increased the number and duration of the cycles.

Build

We used the following program:

Temperature	Time
37 °C	5 min
16 °C	5 min
Go to 1, repeat 29x
60 °C	5 min
4 °C	hold

Test

Unfortunately, the transformation of these GGA products into DH5α cells was unsuccessful, suggesting that the GGA failed.

Learn

We concluded that it may not have been the time of the cycle that was of significance.

Cycle 3

Design

For the next iteration we attempted to change two parameters: the DNA concentration of backbone and fragments and the enzymes in the reaction. Up to this moment we used the NEB GGA enzyme mix for BsaI. For the following attempt, we tested the T4 DNA ligase and the BsaI enzyme separately.

Build

We now used the following protocol:

Reagents	1.	2.	3.	Negative
p15A (70 ng/µL)	0.88 µL	0.88 µL	1.76 µL	1.76 µL
Fragment 1 (50 ng/µL)	2.19 µL	2.19 µL	4.38 µL	-
Fragment 2 (50 ng/µL)	2.10 µL	2.10 µL	4.20 µL	-
Fragment 3 (25 ng/µL)	1.13 µL	1.13 µL	2.26 µL	-
T4 DNA ligase buffer (10 X)	2 µL	2 µL	2 µL	2 µL
NEB GGA Mix BsaI	2 µL	-	2 µL	2 µL
BsaI enzyme	-	1 µL	-	-
T4 DNA ligase	-	1 µL	-	-
ATP (10 mM)	1 µL	1 µL	1 µL	1 µL
MiliQ water	8.7 µL	8.7 µL	2.4 µL	8.7 µL
Total	20 µL	20 µL	20 µL	20 µL

Temperature	Time
37 °C	1 min
16 °C	1 min
Go to 1, repeat 29x
60 °C	5 min
4 °C	hold

We transformed the GGA products into DH5α (three plates per approach). From each plate, we picked 6 colonies for a colony PCR.

Test

For the colony PCR, we expected bands of 900 bp. While some lanes were negative, we had multiple fragments on other lanes (fig. 17-18).

Among many fragments, some were at around 900 bp. We picked these for sequencing. For most of them, the results were negative with exception of 2.3.4. We had positive sequencing results, which suggested a successful GGA reaction. We are currently in the process of investigating this. Due to lack of time we had to end our experiments here. We did not get to the stage of expressing the ferritin.