Engineering
Success
"Some of the best lessons we ever learn we learn from our mistakes and failures. The error of the past is the wisdom and success of the future." - Tryon Edwards
To design and build parts of our biological system, we used engineering principles. In addition, the engineering design cycle was used to tackle challenges in our project. This engineering design cycle consists of four stages Design, Build, Test, and Learn. Nevertheless, we complemented the engineering design cycle with a topic Research, since research is often necessary to successfully design a system. After reflection on the first iteration cycle, one usually starts a new iteration of the cycle from the research step onwards. However, it is possible to enter any stage in a new cycle after reflection. The engineering design cycle is visualized in Figure 1.
The iterations of the design cycle for two aspects of our project are described on this page: cloning strategies and educational lessons for elementary schools. These cycles show how we tackled challenges during our project, what we learned from each engineering iteration, and what we did to improve the design.
Lab
Education
On this engineering success wiki page, we discuss multiple iterations of the DTBL cycle performed for the cloning of one of our plasmids, in which we used different cloning strategies. Each stage of the DBTL cycle is accompanied by an elaborated explanation, including results, thoughts, and decisions.
1.
RESEARCH
In our project, we wanted to determine if antibody-induced GEMS receptor activation is possible. We hypothesized that by introducing an antigen on the EpoR receptor scaffold as an affinity domain, antibodies could activate the receptor. This activation would eventually lead to the expression of our therapeutically relevant protein, interleukin 10 (IL-10) (Figure 1).
DESIGN
Obtaining the DNA that encodes for the longer aa linker length was relatively easy, as these plasmids were already present in the lab storage. One of these plasmids, already including our receptor backbone, coupled to a longer aa linker, was 50-CC (a receptor construct which uses a coiled coil (CC) as affinity domain). This plasmid stated to have a GEMS receptor construct that included an affinity domain that was not of interest to us, however, it was coupled to a 50 aa linker. As this linker length was of interest to
BUILD
Building the new receptor construct with PR3 as an affinity domain with the 50 aa acid linker was expected to be relatively easy. After digestion by the restriction enzymes, an agarose gel was used to separate the digested products (Figure 5). Plasmid length after digestion for 25(GS)_P3 is all around 7000 bp, and PR3 is 663 bp. The bands that are outlined were cut out and DNA was
TEST
To test if the cloning was successful, we transfected the ligated plasmid in TOP10 chemically competent cells. In addition, a negative control was added (only the digested plasmid, no insert) to check for false positives. Transformants were plated on LB-agar plates and grew overnight at 37 ॰C.
LEARN
Taking the sequencing results into account, we hypothesized that the plasmid we started with, did not include the sequence that we expected. Both the anticipated affinity domain and the 50 aa linker were not present in the plasmid.
2.
RESEARCH
As the sequencing results were not as expected, we used commercial Sanger sequencing to determine the original DNA sequence of 25(GS)_P3. From this sequencing of the original plasmid, we concluded that the linker used in this plasmid consisted of 31 amino acids instead of the 50 aa expected. These 31 amino acids are mostly GSS repeats with some other amino acids in between.
DESIGN
Therefore, we had to design a new cloning strategy to avoid the problems which are caused by using BamHI to digest the plasmid. Figure X shows a graphical overview of our new cloning strategy. It makes use of three different restriction sites, EcoRI, BamHI, and NotI, which cleaved the plasmid and PR3 insert in several ways. First of all, the original plasmid was cut with NotI and BamHI, which gives us the plasmid backbone including the ampicillin and kanamycin resistance. Next, the original plasmid was cut with EcoRI and NotI, resulting in the linker coupled to the receptor (without affinity domain). Finally, PR3 is digested using EcoRI and BamHI (Figure 9). If these three parts were ligated correctly, the 31_PR3 plasmid should be obtained.
BUILD
To acquire this 31_PR3 receptor construct, digestions were performed on the original plasmid as described in the cloning strategy above. Digested products were analyzed and separated using an agarose gel (Figure 10). The outlined DNA bands were cut out and purified using a gel purification kit from Qiagen. PR3 was digested with BamHI and EcoRI. Clear, expected bands were seen at 5600 bp (25(GS)_P3 BB), around 1687 bp (EpoR + Linker), and 663 bp (PR3). The outlined DNA bands were cut out and purified.
TEST
To test if the cloning was successful, we transfected the ligated plasmid in TOP10 chemically competent cells. Moreover, a negative control was added (only the digested plasmid, no insert) to check for false positives. To test if the transformations work, a plasmid that we are confident works was inserted as a positive control. Transformants were plated on LB-agar plates and grew overnight at 37 ॰C
LEARN
We repeated all of the digestion and ligation steps, using fresh stocks for every step to ensure that the unsuccessful closing was not affected by any unexpected component in our old stocks. Unfortunately, this did not lead to successful cloning and transformation, as again no cultures were observed.
3.
DESIGN
Unfortunately, the attempted cloning strategy which was discussed in the previous iteration of the DBTL cycle did not succeed. We intended to test an alternative cloning method utilizing a mechanism other than restriction and ligation because this cloning method had failed twice. One cloning method, which is based on a different mechanism, was Gibson assembly. Four new primers were designed (iGEM 016-019), which enabled overlap extension PCR used on the PR3 gBlocksTM gene fragment, and plasmid linearization using PCR of 25(GS)_P3. The latter simultaneously removed the original affinity domain from the 25(GS)_P3 plasmid. The full Gibson assembly strategy is depicted graphically in
BUILD
To obtain the 31_PR3 plasmid using Gibson assembly, two PCR reactions were performed. We performed overlap extension PCR to attach complementary Gibson assembly sites that facilitate the insertion of PR3 into the plasmid. Plasmid linearization was performed to remove the original affinity domain and simultaneously prime the plasmid for Gibson assembly.
TEST
Because we were not able to obtain the full 31_PR3 plasmid using this cloning technique, the “Test” step was omitted in this iteration of the DBTL cycle.
LEARN
Therefore, we discontinued using Gibson assembly to obtain the 31_PR3 plasmid.
4.
DESIGN
Gibson assembly showed to be unsuccessful, depicted by the formation of unexpected PCR products. We had to design a new cloning strategy. Analysis of the 25(GS)_P3 sequence revealed a restriction site encoding for KlfI. As this restriction site is located inside the Igk secretion signal in front of the affinity domain, which is necessary for the transport of the receptor onto the membrane, a new cloning strategy is designed. This new cloning design uses overlap extension PCR to introduce a KlfI restriction site next to the PR3. Moreover, the primer is designed in such a way, that the Igk secretion signal does not get altered
BUILD
Subsequent to receiving the new iGEM 020 primer, an overlap extension PCR reaction on the PR3 gBlock was performed. This should introduce a KlfI site in front of PR3. Hereafter, digestion of the plasmid and gBlocksTM gene fragment was done using the restriction enzymes KlfI and EcoRI. As many of our cloning techniques using PCR were not successful, we wanted to verify if this PCR reaction was successful. An analysis of the samples after PCR was performed using an agarose gel
TEST
Despite the uncertaintly whether the ligation of the first overlap extension PCR was successful, we still wanted to verify if a full plasmid was created. This was done by transforming the ligation product in TOP10 chemically competent cells. Unfortunately, after an overnight incubation step of the transformants, no cultures were seen on the LB-Agar plate.
LEARN
Both the gels used to analyze the digested products after overlap extension PCR and the transformation results suggest that the ligation was not successful. As we used overlap extension PCR to introduce a new restriction site into the gBlocksTM gene fragment, it was expected to see a very clear band around 750 bp as PCR duplicates this DNA exponentially. However, the first gel shows a smear in the lanes of PR3, indicating the presence of many differently-sized PCR products.
5.
DESIGN
Because time was running out to obtain the 31 amino acid receptor construct and wanted to introduce an HA tag next to the original affinity domain, we ordered
BUILD
After receiving and completing the preparation of the new gBlock, the aforementioned cloning strategy is performed. Digestion of the 25(GS)_P3 plasmid and PR3 gBlock was performed using the restriction enzymes KlfI and EcoRI. To validate that the restriction happened correctly and subsequently separate the correct products, digestion products were put on an agarose gel
TEST
To validate if the cloning was successful, ligated products were transformed into TOP10 chemically competent cells. Unfortunately, the transformation was again unsuccessful as no cultures were present on the LB-Agar plate
LEARN
We hypothesize that the unsuccessful cloning can be due to an undigested plasmid. Analysis of the gel seen in Figure 20 (see Build) shows only undigested plasmid. A band at around 270 bp was expected, which is not seen in the gel. This indicates that the plasmid was either not digested or by only one of the two restriction enzymes.
Research
In our project, we wanted to determine if antibody-induced GEMS receptor activation is possible. We hypothesized that by introducing an antigen on the EpoR receptor scaffold as an affinity domain, antibodies could activate the receptor. This activation would eventually lead to the expression of the therapeutically relevant protein, interleukin 10 (IL-10) (Figure 1).
As described on the results and project description wiki pages, we intended to test differently sized amino acids linkers, which were introduced between the antigen (affinity domain) and EpoR receptor scaffold. As it is known that antibodies bind bivalently to antigens which are separated between 30 and 170 Å (with an optimal separation of 160 Å), we sought to create amino acid linker lengths which reside in this range.1 Besides the use of a 0 and 8 amino acid linker, with lengths of 0 Å and 14 Å respectively, the introduction of a longer amino acid (aa) linker would result in a more flexible linker. Click on the word lengths to go the online calculator we used to determine these linker lengths! The calculation is based on the worm-like chain model and empiric observations. We hypothesized that this longer amino acid linker represented the optimal antigen separation for antibody binding more closely. As all three linker variants were already present in plasmids found in the lab storage of the Synthetic Biology research group at our university, the design of new linkers was not necessary. This page will only discuss the cloning strategies for obtaining 31_PR3, therefore, the information on the cloning of the 0 and 8 amino acid linker can be found in our Notebook.
Design
Obtaining the DNA that encodes for the longer 31 aa linker length was relatively easy, as these plasmids were already present in the lab storage. One of these plasmids, already including our receptor backbone, coupled to a longer aa linker, was 50-CC (a receptor construct which uses a coiled coil (CC) as affinity domain). This plasmid stated to have a GEMS receptor construct that included an affinity domain that was not of interest to us, however, it was coupled to a 50 aa linker. As this linker length was of interest to us, we sought to design a cloning strategy applicable to this plasmid. On this wiki page we will refer to this plasmid as 25(GS)_P3. The linker should cosists of 50 amino acids, which was a repeat of 25 times Glycine and Serine (GS). P3 refers to the type of coiled coil which is used as affinity domain.
Although we had access to the plasmid encoding for the 50 amino acid linker, the exact DNA and amino acid sequence were unknown. We hypothesized that the amino acid sequence of this plasmid was consistent with the plasmid encoding for the 8 amino acid linker. We therefore simulated the 50 amino acid linker by multiplying the GS sequence in the 8 amino acid linker (Figure 2).
As stated in the project description, we intended to use these longer linkers between the EpoR part and the proteinase 3 (PR3) as an affinity domain (more explanation in the project description). The design of the PR3 affinity domain was made using a DNA sequence obtained from Uniprot (P24158). The N-terminal of PR3 was adapted to achieve correct folding. Finally, we introduced the restriction sites BamHI and EcoRI together with some extra bases, coding for amino acids, for stability next to the PR3 sequence (Figure 3). This sequence was ordered via IDT as a gBlocksTM gene fragment.
After constructing sequences for both the linker and the PR3 affinity domain, we were able to design a cloning strategy to acquire our receptor scaffold 25_PR3. By digesting both the plasmid and the gBlocksTM gene fragment with BamHI and EcoRI, followed by a ligation reaction using T4 ligase (NEB), the 25_PR3 receptor construct should be acquired (Figure 4).
Build
Building the new receptor construct with PR3 as an affinity domain with the 50 aa linker was expected to be relatively easy. After digestion by the restriction enzymes, an agarose gel was used to separate the digested products (Figure 5). Bands are seen around 7000 kb, which depict the plasmid 25(GS)_P3 after digestion. The bands around 663 bp are the digested PR3. The bands that are outlined were cut out and DNA was purified out of the gel. The digested DNA fragments were stored over the weekend, after which ligation using T4 ligase was performed.
Test
To test if the cloning was successful, we transfected the ligated plasmid in TOP10 chemically competent cells. In addition, a negative control was added (only the digested plasmid, no insert) to check for false positives. Transformants were plated on LB-agar plates and grew overnight at 37 ॰C. The next day, colonies were seen to grow on the plate with the transformants (Figure 6).
To verify that the cloning was successful, the plasmid was sequence verified using commercial Sanger sequencing (BaseClear). The most important sequencing results are shown in Figure 7. The top line, which is partly not filled in with red, represents the missing DNA sequence. This missing DNA sequence would have encoded for the original affinity domain (purple), EcoRI restriction site (black), and the 50 aa linker (gray).
Learn
As seen in Figure 6, both the transformants with ligated plasmid and control transformants plate have grown. Cultures seen on the control plate were not expected to grow, as only the (non-ligated) digested plasmid was transformed in these cells. A possible reasoning for these results could be that one or both of the restriction enzymes were not successful in cleaving the plasmid, leaving the original plasmid unchanged.
Taking the sequencing results into account, we hypothesized that the plasmid we started with, did not include the sequence that we expected. Both the anticipated affinity domain and the 50 aa linker were not present in the plasmid.
Research
As the sequencing results were not as expected, we used commercial Sanger sequencing to determine the original DNA sequence of 25(GS)_P3. From this sequencing of the original plasmid, we concluded that the linker used in this plasmid consisted of 31 amino acids instead of the 50 aa expected. These 31 amino acids are mostly GSS repeats with some other amino acids in between (Figure 8). After analysis of the bases which encoded for this amino acids sequence, multiple BamHI restriction sites were found inside this linker, which is depicted by the underlined amino acids.
Our initial cloning method failed because the BamHI restriction sites appeared more than once in the sequence. The plasmid may be cut at several sites, which after ligation, can produce a variety of ligation products.
Improved design
Therefore, we had to design a new cloning strategy to avoid the problems which are caused by using BamHI to digest the plasmid. Figure 9 shows a graphical overview of our new cloning strategy. It makes use of three different restriction sites, EcoRI, BamHI, and NotI, which cleaved the plasmid and PR3 insert in several ways. First of all, the original plasmid was cut with NotI and BamHI, which gives us the plasmid backbone including the ampicillin and kanamycin resistance. Next, the original plasmid was cut with EcoRI and NotI, resulting in the linker coupled to the receptor (without affinity domain). Finally, PR3 is digested using EcoRI and BamHI (Figure 9). If these three parts were ligated correctly, the 31_PR3 plasmid should be obtained.
Build
To acquire this 31_PR3 receptor construct, digestions were performed on the original plasmid as described in the cloning strategy above. Digested products were analyzed and separated using an agarose gel (Figure 10). The outlined DNA bands were cut out and purified using a gel purification kit from Qiagen. PR3 was digested with BamHI-HF and EcoRI-HF. Clear, expected bands were seen at 5600 bp (25(GS)_P3 BB), around 1687 bp (EpoR + Linker), and 663 bp (PR3). The outlined DNA bands were cut out and purified.
The 3-part ligation was performed using T4 ligase enzyme in a 1:1:3, receptor backbone:plasmid backbone:insert.
Test
To test if the cloning was successful, we transfected the ligated plasmid in TOP10 chemically competent cells. Moreover, a negative control was added (only the digested plasmid, no insert) to check for false positives. To test if the transformations work, a plasmid that we are confident works was inserted as a positive control. Transformants were plated on LB-agar plates and grew overnight at 37 ॰C (Figure 11).
Learn
As seen in Figure 11, the plate containing the negative control and ligated transformants, no cultures could be detected the next day. However, the transformation was effective because cultures grew on the positive control plate. Therefore we hypothesized that either the restriction or ligation step was unsuccessful.
We repeated all of the digestion and ligation steps, using fresh stocks for every step to ensure that the unsuccessful closing was not affected by any unexpected component in our old stocks. Unfortunately, this did not lead to successful cloning and transformation, as again no cultures were observed.
Improved Design
Unfortunately, the attempted cloning strategy which was discussed in the previous iteration of the DBTL cycle did not succeed. We intended to test an alternative cloning method utilizing a mechanism other than restriction and ligation because this cloning method had failed twice. One cloning method, which is based on a different mechanism, was Gibson assembly. Four new primers were designed (iGEM 016-019) , which enabled overlap extension PCR used on the PR3 gBlocksTM gene fragment, and plasmid linearization using PCR of 25(GS)_P3. The latter simultaneously removed the original affinity domain from the 25(GS)_P3 plasmid. The full Gibson assembly strategy is depicted graphically in Figure 12.
Build
To obtain the 31_PR3 plasmid using Gibson assembly, two PCR reactions were performed. We performed overlap extension PCR to attach complementary Gibson assembly sites that facilitate the insertion of PR3 into the plasmid. Plasmid linearization was performed to remove the original affinity domain and simultaneously prime the plasmid for Gibson assembly.
To verify if these PCR reactions were successful, samples of each PCR reaction were examined using an agarose gel (Figure 13). A DNA band are expected at ~6800 bp for the linearized plasmid and ~700 bp for PR3. No band is seen at the expected height for the linearized plasmid, which indicates that our PCR reaction was not successful.
We repeated both PCR reactions, and again analyzed the samples on an agarose gel (Figure 14).
Test
Because we were not able to obtain the full 31_PR3 plasmid using this cloning technique, the “Test” step was omitted in this iteration of the DBTL cycle.
Learn
The gels (Figures 13 and 14) clearly indicate the formation of unintended PCR products during the plasmid linearization step. We hypothesized that the designed Gibson assembly primers have off-target binding sites. However, this assumption could not be confirmed by using in-silico methods to discover off-target binding sites.
Therefore, we discontinued using Gibson assembly to obtain the 31_PR3 plasmid.
Improved Design
Gibson assembly showed to be unsuccessful, depicted by the formation of unexpected PCR products. We had to design a new cloning strategy. Analysis of the 25(GS)_P3 sequence revealed a restriction site encoding for Klf1. As this restriction site is located inside the Igk secretion signal in front of the affinity domain, which is necessary for the transport of the receptor onto the membrane, a new cloning strategy is designed. This new cloning design uses overlap extension PCR to introduce a Klf1 restriction site next to the PR3. Moreover, the primer is designed in such a way, that the Igk secretion signal does not get altered (Figure 15).
Build
Subsequent to receiving the new iGEM 020 primer, an overlap extension PCR reaction on the PR3 gBlock was performed. This should introduce a Klf1 site in front of PR3. Hereafter, digestion of the plasmid and gBlocksTM gene fragment was done using the restriction enzymes Klf1 and EcoRI-HF. As many of our cloning techniques using PCR were not successful, we wanted to verify if this PCR reaction was successful. An analysis of the samples after PCR was performed using an agarose gel (Figure 16).
A clear band is seen at 6700 bp for linearized 25(GS)_P3. A very big smear is seen for the digested PR3 (expected 663 bp). As we were unsure if the band indicated a successful PCR reaction, extracted the DNA from this gel. An overnight ligation reaction was performed, resulting in the formation of 31_PR3. As it was uncertain whether the ligation worked, we repeated the overlap extension PCR followed by digestion by Klf1 and EcoRI-HF and analyzed the results again using an agarose gel (Figure 17).
Test
Despite the uncertaintly whether the ligation of the first overlap extension PCR was successful, we still wanted to verify if a full plasmid was created. This was done by transforming the ligation product in TOP10 chemically competent cells. Unfortunately, after an overnight incubation step of the transformants, no cultures were seen on the LB-Agar plate.
Learn
Both the gels used to analyze the digested products after overlap extension PCR and the transformation results suggest that the ligation was not successful. As we used overlap extension PCR to introduce a new restriction site into the gBlocksTM gene fragment, it was expected to see a very clear band around 750 bp as PCR duplicates this DNA exponentially. However, the first gel shows a smear in the lanes of PR3, indicating the presence of many differently-sized PCR products.
The second PCR reaction results, shown in Figure 17 (see Build), were not successful, as no bands can be seen in the wells of PR3.
As we wanted to include an HA-tag in our receptor design as shown in Figure 18 (see Build), we needed to switch again from this cloning design to another.
Improved Design
Because time was running out to obtain the 31 amino acid receptor construct and wanted to introduce an HA tag next to the original affinity domain, we ordered a new PR3 gBlocksTM gene fragment with an attached HA-tag. It also included the Klf1 tag including the part of the Igk secretion signal as described in our last cloning method. The cloning method we wanted to use is similar to the previous method, however, gBlocksTM gene fragments were ordered to obviate the overlap extension PCR step (Figure 19). This simplifies this cloning method to only a 2-part restriction and ligation cloning.
Build
After receiving and completing the preparation of the new gBlock, the aforementioned cloning strategy is performed. Digestion of the 25(GS)_P3 plasmid and PR3 gBlock was performed using the restriction enzymes Klf1 and EcoRI. To validate that the restriction happened correctly and subsequently separate the correct products, digestion products were put on an agarose gel (Figure 20).
Outlined DNA was purified (Qiagen gel extraction kit), and ligated using T4 ligase (overnight at 4 ॰C). This should provide us with the 31_HA_PR3 plasmid.
Test
To validate if the cloning was successful, ligated products were transformed into TOP10 chemically competent cells. Unfortunately, the transformation was again unsuccessful as no cultures were present on the LB-Agar plate (Figure 21).
Learn
We hypothesize that the unsuccessful cloning can be due to an undigested plasmid. Analysis of the gel seen in Figure 20 (see Build) shows only undigested plasmid. A band at around 270 bp was expected, which is not seen in the gel. This indicates that the plasmid was either not digested or by only one of the two restriction enzymes.
Conclusion
We have shown 5 iterations of the DBTL cycles, which describe our process to obtain the 31_PR3 receptor scaffold. Unfortunately, even after all the cloning attempts which were based on different mechanisms, we were not able to successfully clone the affinity domain PR3 in the plasmid 25(GS)_P3.
However, because obtaining a receptor scaffold including a longer amino acid linker is still of high importance for the future of !MPACT, we will end this DBTL cycle with suggestions that could result in the successful cloning of a longer linker length in a receptor construct.
We hypothesize that using Gibson assembly cloning to obtain 31_PR3 has the most potential to succeed, as by using this technique you have complete control over what parts are inserted. By designing new Gibson primers specific for 25(GS)_P3 and ensuring they do not have off-target binding sites, we expect that the cloning could be done relatively easily.
If problems still arise while using this method, other methods could include the use of a different starting plasmid (e.g. 4_P3). By using plasmid linearization with primers designed to remove the full affinity domain and original linker, a plasmid including the receptor backbone can be obtained. Next, a gBlocksTM gene fragment which encodes for the affinity domain of choice, coupled to the linker length of choice, can be ordered. Next, both fragments can be ligated togheter, resulting in the receptor construct with a longer linker length. If all fails, you could still contact IDT or Twist to synthesize a gene or even use their cloning service.
- Shaw A, Hoffecker IT, Smyrlaki I, et al. Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies. Nat Nanotechnol. 2019;14(2):184-190. doi:10.1038/s41565-018-0336-3
In addition to our project, we focused on educating different target groups about synthetic biology. One of these target groups was elementary school children. For these children, we organized a DNA lesson, given at the elementary school in Haaren. More information about our experience and the lesson can be found on the education page.
1.
RESEARCH
Before we could start designing, more information was required. Such as, the age of the children and what do they already know about DNA. We asked the elementary school teacher for this information and learned the children would be about 10 to 11 years old. Furthermore, the school was in the middle of a project about DNA. Therefore, the students had already a bit of knowledge about DNA. This was implemented into the design.
DESIGN
The teachers of these classes wanted a lesson about what DNA is, and what research you can do with DNA. This was an addition to a running project the school had; the teachers were looking for experts to tell them about synthetic biology. Therefore, the purpose of this lesson was to make them familiar with synthetic biology starting with the principles of DNA.
BUILD
The presentation was separated into two parts. The first part contained general information about DNA. Explained with the use of lots of simple icons. The second part explained the research which can be done with DNA. This was explained by using simple examples and again icons. The parts were separated by a small, interactive game, the DNA game, where the students could form a DNA sequence presented on the presentation with our pre-prepared nucleotide letter signs. We made signs with a cord with the letters A, T, G, and C (the nucleotides) which the students could use to form a DNA sequence of four nucleotide pairs.
TEST
Before performing the lesson on the 16th of May in Haaren, we tested it a couple of times. Doing so by presenting for each other and making sure we spoke in an easy and clear language. The real test was performed on the 16th of May for the students and teacher. We gave the presentation, explained everything clearly and understandable to the students, and the students played the game. We received a ton of questions from the children! Not only about the information we gave, but also questions that were built on the information we gave. So, we could conclude that they did understand everything we explained and wanted to learn even more about DNA.
LEARN
After this lesson, we evaluated with our team, and we also received some feedback from the teacher. We learned that we handled the questions from the children well and were adapting to the level of the elementary school children. Furthermore, it was nice that there was a lot of time for questions. Although, when answering questions, we could say ’I do not know’ if we do not know the answer to a question.
2.
RESEARCH
The next lesson would be given to the exact same age group as before, only at a different school. This time we would give our lesson in Uden to teach children about DNA. These children did not have any prior knowledge about DNA.
DESIGN
The design for the setup of this lesson is mostly the same, as there was no negative feedback about the presentation and information. Only the execution of the presentation and the game did change, taking in the points of improvement from the teacher as mentioned above.
BUILD
The alterations in design were practiced by practicing the presentation again.
TEST
We performed the next lesson on the 20th of June in Uden. This time we made sure enough time was taken to explain everything clearly, since these children did not have prior knowledge. Besides, we implemented the feedback by explaining the game step by step and asking children one by one to the front of the classroom. Furthermore, when answering questions, we took a lot of time. This is the same as the previous time, since this was seen as very positive. Now, we also said that we did not know how to answer a question when we did not know.
LEARN
After this lesson we reflected again. We could happily conclude all the previous feedback points were implemented. From the teacher we received some new (positive) feedback as well. She mentioned the presentation had a clear structure and visualized our words. There was a lot of interaction with the children. Furthermore, she gave one tip for improvement. She mentioned it is a good idea to let the children do something themselves. Although she also mentioned that she enjoyed the interactive exercise we had thought about, there the children did something themselves as well.
Research
Before we could start designing, more information was required. Such as, the age of the children and what do they already know about DNA. We asked the elementary school teacher for this information and learned the children would be about 10 to 11 years old. Furthermore, the school was in the middle of a project about DNA. Therefore, the students had already a bit of knowledge about DNA. This was implemented into the design.
Design
The teachers of these classes wanted a lesson about what DNA is, and what research you can do with DNA. This was an addition to a running project the school had; the teachers were looking for experts to tell them about synthetic biology. Therefore, the purpose of this lesson was to make them familiar with synthetic biology starting with the principles of DNA.
After receiving the requirements of the teachers regarding the content of the lesson we started brainstorming. The proposed design is a presentation with a lot of easy and explaining images and an interactive game. With the game, the students would form their own short DNA sequence, by standing at the front of the class with a sign of a nucleotide around their neck and holding hands.
Build
The presentation was separated into two parts. The first part contained general information about DNA. Explained with the use of lots of simple icons. The second part explained the research which can be done with DNA. This was explained by using simple examples and again icons. The parts were separated by a small, interactive game, the DNA game, where the students could form a DNA sequence presented on the presentation with our pre-prepared nucleotide letter signs. We made signs with a cord with the letters A, T, G, and C (the nucleotides) which the students could use to form a DNA sequence of four nucleotide pairs.
Test
Before performing the lesson on the 16th of May in Haaren, we tested it a couple of times. Doing so by presenting for each other and making sure we spoke in an easy and clear language. The real test was performed on the 16th of May for the students and teacher. We gave the presentation, explained everything clearly and understandable to the students, and the students played the game.
We received a ton of questions from the children! Not only about the information we gave, but also questions that were built on the information we gave. So, we could conclude that they did understand everything we explained and wanted to learn even more about DNA.
Learn
After this lesson, we evaluated with our team, and we also received some feedback from the teacher. We learned that we handled the questions from the children well and were adapting to the level of the elementary school children. Furthermore, it was nice that there was a lot of time for questions. Although, when answering questions, we could say ’I do not know’ if we do not know the answer to a question.
Also, the teacher mentioned the game was a nice active manner of teaching the principles of DNA. Although we should explain the game easier for the next group. Also, it might work better if we would choose one child at a time when bringing the children to the front of the class for the game. The teacher also mentioned that this DNA lesson was a good addition to the knowledge that the students already had. We had a powerful attitude in front of the class and were enthusiastic about the subject. Although, the next time we should talk slower.
Research
The next lesson would be given to the exact same age group as before, only at a different school. This time we would give our lesson in Uden to teach children about DNA. These children did not have any prior knowledge about DNA.
Design
The design for the setup of this lesson is mostly the same, as there was no negative feedback about the presentation and information. Only the execution of the presentation and the game did change, taking in the points of improvement from the teacher as mentioned above.
The plan is when the game starts to ask children one by one to the front of the class and take more time to explain the game step by step. Furthermore, when answering questions, we planned on taking enough time to answer them, and if we do not know the answer, we should say it.
Build
The alterations in design were practiced by practicing the presentation again.
Test
We performed the next lesson on the 20th of June in Uden. This time we made sure enough time was taken to explain everything clearly, since these children did not have prior knowledge. Besides, we implemented the feedback by explaining the game step by step and asking children one by one to the front of the classroom. Furthermore, when answering questions, we took a lot of time. This is the same as the previous time, since this was seen as very positive. Now, we also said that we did not know how to answer a question when we did not know.
We gave the lesson with the same enthusiasm as the previous time. Also, this time the lesson was received positively, again the children asked tons of questions.
Learn
After this lesson, we evaluated with our team, and we also received some feedback from the teacher. We learned that we handled the questions from the children well and were adapting to the level of the elementary school children. Furthermore, it was nice that there was a lot of time for questions. Although, when answering questions, we could say ’I do not know’ if we do not know the answer to a question.
Also, the teacher mentioned the game was a nice active manner of teaching the principles of DNA. Although we should explain the game easier for the next group. Also, it might work better if we would choose one child at a time when bringing the children to the front of the class for the game. The teacher also mentioned that this DNA lesson was a good addition to the knowledge that the students already had. We had a powerful attitude in front of the class and were enthusiastic about the subject. Although, the next time we should talk slower.