Engineering

Here you can read about the engineering cycles our project and project parts have been through.

Engineering Cycle for Project Design

1. Design

We have made clear plans for the project and divided our project into three parts that we would focus on. Library construction, Ribosome Display and Bioreporter building. For each part we prepared by reading literature of what has already been done, and started planning on what we could do differently. We also asked a lot of help from our instructors and mentors who have the best knowledge in each subject, to ensure that we avoid any unnecessary mistakes.

2. Build

After designing the plan for each part, we will begin composing the actual plasmid, or DARPin design for the testing phase. We ordered all designed fragment parts and on the dry lab side, we built the algorithms needed for the selection of DARPins. We based our construction on the help we had gotten in the design phase, and got inspired for the building phase from what we had been reading from the literature ( Stumpp et al., 2008, Team IISER, Kolkata).

3. Test

During testing we will do most of the lab work, where we will test how well our designed and built fragments actually works. We will both do drylab and wet lab work during this time to optimise the time spent. We worked simultaneously on the different parts of the project.

4. Learn

From the results in the testing phase, we will then reconsider our constructs and if needed go back to the designing phase to see if we need to change something in the construct or if the issues could possibly be fixed with outer changes, especially in the wet lab work.

Our Project in a Nutshell

Our project is based on chronic wounds and the build-up of biofilm, which is a both local and a global issue our iGEM team wants to tackle. Our solution is based on protein-peptide binding modelling and execution and for that, we also need tools to confirm that our design actually works. For this, we needed to design both the binding protein (DARPin) as well as a bioreporter for detection. We needed a bioreporter that would give us something to measure the activity of specific genes when our target peptide was both present and absent. For this, we planned based on the research and based on the past iGEM team's success to do similar parts that were found in the parts registry. We thought that measuring GFP would be the most manageable signal to read for us, so we planned to have one inducible promoter that would start transcription of the fluorescent proteins only if the target peptide was present. We would also have the required signalling pathways proteins expressed at all times. For ordering all parts, we thought we would order as big sequence fragments as possible to minimise the time needed for cloning. We planned to use basic restriction cloning for both parts.
In the following sections we describe how we used the engineering cycles to work on these parts.

Cycle 1

1. Design

We needed a bioreporter that would give us something to measure the activity of specific genes when our target peptide was both present and absent. For this, we planned based on the research and based on the past iGEM team's success to do similar parts that were found in the parts registry. We thought that measuring GFP would be the most manageable signal to read for us, so we planned to have one inducible promoter that would start transcription of the fluorescent proteins only if the target peptide was present. We would also have the required signalling pathways proteins expressed at all times. For ordering all parts, we thought we would order as big sequence fragments as possible to minimise the time needed for cloning. We planned to use basic restriction cloning for both parts.

So, for our bioreporter, we needed to make 2 composite parts which included all the needed parts to complete our bioreporter. To learn more about the individual parts and constructs, see Parts (link) page. Our part1 for the bioreporter consists of two promoters in two different directions. The idea of the bidirectional promoters was from past year's iGEM team, especially team IISER Kolkata, who had done a similar construct in 2021. They had proved that the bidirectional model of the two promoters worked better than if the promoters were to be placed after each other. After both promoters, there is the RBS sequence.

2. Build

We tried to order this part1 (fig.1) from IDT, however, in the production phase, we ran into the problem of the part not passing the quality control, even though the part was accepted in the initial check before ordering.

Sequence for first draft of bioreporter.
Figure 1. First draft of Part1 for bioreporter.

We got in contact with our sales representative Silvija Jovic from IDT, with whom we had a meeting about our project. We discussed the issues with her and agreed on sending the fragment to the IDT specialists who made some changes to the beginning of the sequence. The issue with the fragment sequence was the T nucleotide heavy beginning which made the sequence probably very unstable and that is why it failed the quality control tests. In fig. 2, the change to the first sequence is shown in red colour.

Sequence for second draft of bioreporter.
Figure 2. Second draft of Part1 for bioreporter.

3. Test

We started working first with the second draft of the fragment. In the testing phase for the second draft of part1, we succeeded up to the transformation phase. In Figure 3. Is the successfully amplified part1 (second draft).

Gel picture
Figure 3. The DNA concentration of the band from the annealing temperature 62 °C and 63 °C was measured at 17.5 ng/µL with a purity at A260/280 of 1.95 and the the band from the annealing temperature 68 °C was measured at 10.5 ng/µL with a purity at A260/280 of 1.42.

After this we continued the digestion and ligation. The digestion gel picture is presented in Figure 4.

Gel picture
Figure 4. The digest of part1 and pET28a-sfGFP plasmid containing BBa_K4159009. The DNA concentrations were measured at 11.8 ng/µL for part1 and 3.1 ng/µL for pET28a-sfGFP plasmid containing BBa_K4159009.

However, for some reason, the transformation of the fragment did not work. We tried different cells for the transformation, both alpha5 and top10 E.coli cells for the transformation, but with zero success in getting colonies. We also tried to prolong the ligation time in case the issue was there, and different ratios (3:1 and 5:1) of DNA to vector ratio. We also went back to the first step of PCR, and tried different annealing temperatures to optimise it to have as high of a concentration of the fragment for digestion and ligation as possible.

4. Learn

During the time we were doing the lab work on this, we were also taking part in the Nordic iGEM Conference in Sweden. During the presentations at the conference, we got some feedback about our parts for the bioreporter. The concern that there was too little space between the promoters was raised, and it made us think that the small space in between could result in neither of the promoters working. We hypothesised that the lack of space could disrupt both promoters as there was not enough room, specifically for the P2 promoter to be activated. We discussed this also with our mentors and got the advice from Sami Jalil to try the Second draft part1 first, and in case it doesn't work, design a third with about 20-50bp in between the promoters.

Cycle 2

1. Design

After, struggling and troubleshooting in the lab with the second draft, we went back to the design phase again.
For our third design we removed the 6bp spacer from the second draft and replaced it with a random sequence of 50bp. The random sequence was generated from the University of California's Random DNA generator. The GC content for the randomization was put as 0.5.

Sequence for third draft of bioreporter.
Figure 5. Third draft of Part1 for bioreporter.

2. Build

We hoped that the change in spacer length would be the fix to our problems. The difference from the earlier fragment was also the supplier. As our IDT account had reached its limit, we ordered now from Twist Bioscience, however, this should not change anything in the protocols.

3. Test

For the testing phase we followed the same protocol as before in the beginning. We had some troubles in the beginning with the PCR and got very low concentration after the amplification, however, at the end we succeeded again all the way to the transformation phase. After a few trials and failure of the transformation with the new third draft of the part1 fragment, we decided to change the protocol a bit and lowered the temperature and decreased the time of the heat shock from 45C in 45 sec to 42C for 30sec.

4. Learn

This change in protocol gave a positive result and the plasmid was successfully transformed into the cells. We assume that the change in protocol had in this case the bigger effect on the successful outcome. However, we think it was still a good idea to change the length of the spacer in between of the promoters to avoid that they would somehow falsely start transcribing the signal even if there is no signalling molecule binding to the cell. The temperature change in the transformation protocol was also a good lesson to learn, that it might not always be good to have the temperature so high, even if the cells used in the transformation are supposed to handle the temperature in the transformation.

References

  1. Stumpp, M. T., Binz, H. K., & Amstutz, P. (2008)
    DARPins: a new generation of protein therapeutics
    Drug discovery today, 13(15-16), 695-701
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