Overview
Overview
During the Wet Lab-phase of a project, each experiment is a unique journey for every iGEM team. However, these journeys are hardly ever
straightforward. iGEM members come across countless challenges, during which, identifying the actual problems and crafting solutions is essential
but also insightful.
The iGEM cycle was the guiding compass for us, as it presents a structured path to tackle these challenges: Design, Build, Test, Learn.
In this narrative, we share our own iGEM experience, discussing the problems we tackled as a team.
Choosing the right vectors
Choosing the right vectors
Design
Selecting the appropriate vectors for our constructs proved to be a more challenging task than we initially anticipated.
In order to make our experiments efficient and reduce their complexity, we needed to decide upon a selection marker that best fits our project.
We ended up designing our constructs based on vectors with different antibiotic resistance.
Additionally, considering our references, we needed to take into account that one of our constructs was going to be based on a low-copy number plasmid while the others, on high-copy number plasmids.
Specifically, the plasmid vectors employed by our references were:
pBR322: A low-copy number, ampicillin and tetracycline resistant plasmid utilized for the moderate overexpression of hemD gene, providing the best yield in the accumulation of 5-ALA [1].
pRSFDuet-1:A high-copy number, ampicillin resistant plasmid used for the co-overexpression of hemL, hemAs, hemF, that gives the best yield in 5-ALA accumulation when combined with pBR322’s moderate overexpression of hemD. [1]
pOSIP-TT: A classic tetracycline resistant plasmid vector usually employed in clonetegration techniques. [2]
pETDuet-1: A high-copy number, ampicillin resistant plasmid, carrying two T7 Promoters, making it an ideal vector choice for the expression of rhtA and Human Catalase, two genes we wanted to co-express under the regulation of two different promoters. [3]
Due to the fact that some of these plasmids exhibit resistance to the same antibiotics, we needed to employ alternative vectors, with diverse antibiotic resistance profiles, always taking into account that one had to be a low-copy number, while the others had to be high-copy number plasmids.
The final vectors we decided to employ for building our constructs were:
pBBR1MCS-3: A low copy number vector, tetracycline resistant, chosen for the moderate overexpression of hemD. [4]
pET-29c(+): A high copy number plasmid, kanamycin resistant for the overexpression of hemL, hemAs and hemF.[5]
pETDuet-1: A high-copy number, ampicillin resistant plasmid for the co-expression of rhtA exporter and human catalase genes.[3]
pOSIP-CH: An alternative plasmid vector for the clonetegration of Rluc8 gene, chloramphenicol resistant. [6]
Build
In order to build our constructs, we initially had to isolate our plasmids and conduct numerous experiments,
including diagnostic digestions and self-ligation reactions to assess the effectiveness of our double digestions.
Although we had to change our strategy and chose alternative vectors for our design, we always kept in mind that
we needed our plasmids to have different antibiotic resistance.
Test
We managed to build some of our constructs using Hi-Fi DNA Assembly. One of the ways we tested our assembly results was
antibiotic treatment. Additionally, the variety of antibiotic resistances proved valuable in our troubleshooting experiments.
Detecting the presence of our vectors throughout our wet lab experiments was a straightforward and dependable process.
Learn
During this process, we came to understand the importance of adaptability in a project like ours, where we may need to make
design adjustments to suit our needs and available resources. We learned to always be stand-by in order to reconsider our project
design, while always maintaining the initial purpose of every construct. We also realized that one change in our project design
can trigger a domino effect, making it necessary to change other aspects of our project too.
The selection of our plasmid vectors based on antibiotic resistance would also play a pivotal role in our upcoming experiments, including the transformation into our final bacterial strain BL21 and verifying whether our bacteria have successfully taken up all of our plasmids.
The selection of our plasmid vectors based on antibiotic resistance would also play a pivotal role in our upcoming experiments, including the transformation into our final bacterial strain BL21 and verifying whether our bacteria have successfully taken up all of our plasmids.
Building our constructs
Building our constructs
Construct 1
Design
Following the optimization of the heme biosynthesis pathway method [1], a low copy- number vector for moderate overexpression of hemD was required. We didn’t have access to using pBR322 but instead we found pBBR1MCS-3 from our PI’s collaborator laboratory. pBBR1MCS-3 had all the necessary characteristics for our purpose.Therefore, our initial design was based on pBBR1MCS-3:
Vector
pBBR1MCS-3Gene inserted
hemD
This vector features a ColE1/pMB1/pBR322/pUC origin of replication and it is tetracycline resistant.
pBBR1MCS-3 vector
While pBBR1MCS-3 features a T7 promoter, it lacks both an RBS (Ribosome Binding Site) and a T7 terminator. To ensure the inclusion of these crucial regulatory elements, we incorporated the RBS and T7 terminator from pETDuet-1 into our insert. Our insert construction proceeded as follows:
Initial hemD insert
The vector arrived in bacterial glycerol stocks, and we initiated our experiments with plasmid isolation. Unfortunately, our initial attempt to isolate pBBR1MCS-3 was unsuccessful, but we attributed it to the fact that low-copy-number plasmid isolation is already a challenge. Following a second unsuccessful attempt, we began to question what might have been causing the issue.
We then began a series of troubleshooting experiments in order to isolate our vector.
pBBR1MCS-3 troubleshooting experiments PDF HERE
Despite exploring various alternative methods, our efforts to isolate this plasmid remained unsuccessful. Consequently, we made the decision to modify our design and identified pTU-2 as a suitable alternative vector.
Construct 1 - Final Design
Construct 1 - pTU2-A-RFP (p15A origin)/ hemD
hemD insert
Build
We attempted to build our final construct using Hi-Fi DNA assembly and designing the appropriate set
of primers to incorporate the required overlapping regions into the hemD insert.
Test
We achieved successful assembly of our construct, as we observed colonies in our transformation plates post-assembly
.To validate our results, we also performed colony PCR and diagnostic digestions.
Construct 1 transformation plate after assembly
Learn
During this troubleshooting process, we learned a lot about low copy plasmid isolation, the role of chloramphenicol in liquid bacteria cell cultures and the
impact of using various antibiotic volumes on improving the efficacy of our experiments.
Construct 2
Design
Our objective was to overexpress the hemL, hemAs, and hemF genes under two separate T7 promoters. To achieve this, we needed to identify a high-copy number plasmid that carries both the T7 promoters and the associated regulatory elements.Our initial reference recommended that we use the pRSFDuet-1 vector, but unfortunately, we did not have access to that particular plasmid. Instead, we came across the pET-29c(+) vector, which is a high-copy number plasmid, although containing only one set of the essential regulatory elements: one T7 promoter, one RBS and one T7 terminator. It was our responsibility to introduce the second set of these elements into our insert.
When planning the design of construct 2, our initial approach involved incorporating all three genes, along with the extra T7 promoter, RBS, T7 terminator, and tags, into a single insert. For this purpose, we created a 3789 bp insert and designed only one set of primers.
Initial hemL, hemAs, and hemF
However, we were not able to order a gene fragment of that extended length through our iGEM sponsorships. Consequently, we made the decision to divide our insert into three distinct fragments, which would later be assembled as a single unit by utilizing the appropriate overlapping regions.
Designing the correct primers for this assembly was definitely a challenge. We needed to create primer pairs that would introduce the right overlap regions to our inserts, enabling them to seamlessly join together during our assembly process and mimic a single continuous insert. Our final construct was designed as follows:
pET-29c(+)
Build
We attempted to build our construct 2 by combining all three inserts into a single Hi-Fi DNA assembly reaction, using three different pairs of primers
in order to add the necessary overlap regions at first in all of our inserts.
Test
After the assembly reaction, we performed a colony PCR in order to verify our results. We picked 5 different colonies from the
transformation plates we had prepared after the assembly and employed the Forward primer for the hemL and the Reverse primer for
the hemF insert. These primers would give us a PCR product of 3975bp, including all three inserts (hemL, hemAs, hemF).
At first, we thought that the assembly did not work because we did not get the expected results from our colony PCR. In fact, we didn’t observe any PCR products for none of the 4 colonies we chose in our Gel Electrophoresis. We then performed diagnostic digestions for the colonies 1,2& 4 with SalI, an enzyme that has two cutting sites in our region of interest, therefore giving us a 1919bp & a 7182bp fragment. We also performed a digestion with HindIII, a restriction enzyme that gives a 3.530bp and a 5571bp fragment. We did not observe the expected DNA fragments In our Gel Electrophoresis after the digestions. Both of our Electrophoresis results are shown below:
At first, we thought that the assembly did not work because we did not get the expected results from our colony PCR. In fact, we didn’t observe any PCR products for none of the 4 colonies we chose in our Gel Electrophoresis. We then performed diagnostic digestions for the colonies 1,2& 4 with SalI, an enzyme that has two cutting sites in our region of interest, therefore giving us a 1919bp & a 7182bp fragment. We also performed a digestion with HindIII, a restriction enzyme that gives a 3.530bp and a 5571bp fragment. We did not observe the expected DNA fragments In our Gel Electrophoresis after the digestions. Both of our Electrophoresis results are shown below:
Gel Electrophoresis after diagnostic digestions
Gel Electrophoresis after colony PCR
Learn
Through this process we realized that performing a colony PCR for such a long insert carried significant risks.
Furthermore, we found ourselves wondering about the reasons behind the possible failure of our assembly.
After discussing the issue with our PI and instructor, we came to a conclusion that assembling a construct with 3 different inserts like ours is not always easy. Many things may have led to the assembly failure and the most important one is that these inserts have not been assembled like this before and we can’t be sure about them functioning well. In addition, mistakes in the primer design and the strategy of our assembly are possible and may have caused the issue for this construct. Last but not least, our decision to rely on just 5 colonies for post-assembly PCR was questioned, as this number didn't provide a representative sample of the entire reaction.
However, given the abundance of colonies on our transformation plate and our prior examination of vector self-ligation, which indicated a low likelihood of such events, we decided to attempt another colony PCR reaction. This time, we prepared 10 samples, each one consisting of 5 colonies from our plate. For this PCR, we used both the Forward and Reverse primers for the hemAs insert (1416bp), as it occupies the middle insert in our construct.
We believed that if the hemAs insert could be successfully amplified by PCR, it would signify that our other two inserts had been successfully assembled into our vector as well, given the reliability and specificity of Hi-Fi DNA Assembly. Our Gel Electrophoresis results confirmed the successful assembly of the hemAs insert.. We faced an issue with our DNA ladder, which was not loaded on the gel, but comparing our results to our controls and other PCRs we have conducted for hemAs, we firmly believe that this is the right insert being observed.
Furthermore, we found ourselves wondering about the reasons behind the possible failure of our assembly.
After discussing the issue with our PI and instructor, we came to a conclusion that assembling a construct with 3 different inserts like ours is not always easy. Many things may have led to the assembly failure and the most important one is that these inserts have not been assembled like this before and we can’t be sure about them functioning well. In addition, mistakes in the primer design and the strategy of our assembly are possible and may have caused the issue for this construct. Last but not least, our decision to rely on just 5 colonies for post-assembly PCR was questioned, as this number didn't provide a representative sample of the entire reaction.
However, given the abundance of colonies on our transformation plate and our prior examination of vector self-ligation, which indicated a low likelihood of such events, we decided to attempt another colony PCR reaction. This time, we prepared 10 samples, each one consisting of 5 colonies from our plate. For this PCR, we used both the Forward and Reverse primers for the hemAs insert (1416bp), as it occupies the middle insert in our construct.
Construct 2 transformation plate after assembly
We believed that if the hemAs insert could be successfully amplified by PCR, it would signify that our other two inserts had been successfully assembled into our vector as well, given the reliability and specificity of Hi-Fi DNA Assembly. Our Gel Electrophoresis results confirmed the successful assembly of the hemAs insert.. We faced an issue with our DNA ladder, which was not loaded on the gel, but comparing our results to our controls and other PCRs we have conducted for hemAs, we firmly believe that this is the right insert being observed.
Final PCR
Construct 3
Design
In construct 3 we wanted to incorporate rhtA and Human catalase gene under the regulation of two different T7 promoters. Since our vector already carried the two promoters, we needed to perform two separate cloning steps in order to build our construct.When designing our construct our initial plan involved using the EcoRV restriction enzyme for the first cloning step to introduce the catalase gene. Following that, in the second cloning step, we implemented the SacI enzyme to insert the rhtA gene.
The final construct deriving from this strategy was the following:
Initial cloning strategy- Catalase
Initial cloning strategy- Catalase
In this figure, we can see that Catalase was initially inserted after digestion with EcoRV. In the left side of the figure, you can see the first bases of the Catalase Insert following after the 5’-GAT-3’ sequence and in the right side of the figure, you can see the last bases of the Catalase Insert followed by the sequence 5’-ATC-3’. These two sequences are specifically because of EcoRV, as they represent the blunt edges left after digesting with this enzyme.EcoRV’s sequence of recognition is:
EcoRV’s sequence
This strategy exhibits one major disadvantage: The above order of the two cloning steps must be strictly followed, otherwise, our construct can not be assembled properly. This issue arises due to the presence of a cutting site for the EcoRV restriction enzyme within the rhtA gene, making it unsuitable for use in the second cloning step.
This problem led us to reconsider the design of our construct. Due to our limited time for experimentation and the goal of completing our construct assembly by the end of the competition, we opted to simultaneously pursue both cloning processes. This strategy ensured that if one of the cloning attempts encountered initial challenges, we would have the alternative of the other cloning process to proceed with our assembly. To achieve this, we needed to switch the restriction enzyme used to cut the plasmid for the insertion of the catalase gene.
We decided to use BglII instead of EcoRV for our catalase cloning, a restriction enzyme that does not present a cutting site inside the rhtA insert. As a result, we had to reconsider the design of our construct and primers to make the double cloning process feasible. The final construct was designed as follows:
Construct 3 overview
Build
We attempted to build our final construct 3 using Hi-Fi DNA assembly and designing the appropriate set of primers to
incorporate the required overlapping regions into our inserts.
Test
After analyzing the results of our colony PCR and conducting diagnostic digestions with restriction enzymes, we came to the
conclusion that both of our cloning steps were successful. In addition, the transformation results after our assembly for each
insert confirmed this hypothesis.
Construct 3 (rhtA) transformation plate after assembly
Construct 3 (Catalase) transformation plate after assembly
Learn
Despite being proven as unnecessary, this process provided us with important lessons. For example, we learned how to anticipate and proactively make adjustments to avoid schedule disruptions. The redefinement of our project's design would serve as a valuable asset in case any issues arose during our cloning procedures,
allowing us to address this challenge without the need for a complete redesign.
Construct 4
Design
Given that our other vectors displayed resistance to various antibiotics, we decided to employ a similar approach for our clonetegration plasmid too. Since we already had vectors resistant to ampicillin, tetracycline, and kanamycin, we selected pOSIP-CH, a vector with chloramphenicol resistance.We designed our Rluc8 clonetegration construct as follows:
Rluc8 clonetegration construct
Due to the modification in our hemD construct 1 design, where we switched from pBBR1MCS-3 to pTU2-A-RFP (p15A origin) as our final vector, we ended up having two plasmids with the same antibiotic resistance. We then decided to revise our clonetegration constructs design by incorporating pOSIP-TT (tetracycline-resistant) as our delivery vector.
Vector
pOSIP-TT (P21)The updated construct was designed as follows:
Construct 4 overview
Build
In order to build our clonetegration construct, we first needed to replicate and isolate pOSIP-TT. This plasmid was
included in our iGEM 2023 Distribution Kit, but we only had 1-2 nanograms available for our experiments.
Since we needed at least 50 nanograms of our vector for cloning, we decided that the best way to increase our
plasmid’s volume was by transforming it into DB3.1 bacterial cells, a strain resistant to the ccdB toxin expressed by pOSIP-TT.
Test
DB3.1 cells were sent to us from our PI’s collaborative laboratory and we began our transformation experiments.
After reviewing our initial results, we noticed an issue with our tetracycline plates.
This led us to initiate a second round of troubleshooting experiments related to our project,
only to find that the DB3.1 strains we had received were resistant to tetracycline.
Learn
Once again, through our second troubleshooting round we had the opportunity to explore and gain valuable experience
in addressing several challenges and conducting the right experiments to prove a hypothesis. Additionally, we
learned the importance of always checking the reagents received from external laboratories to ensure they align precisely
with our requests.
Designing our primers
Designing our primers
Design
Primer design was also quite demanding. We needed to create the correct primers in order for our constructs to be assembled right.
We had to ensure the precise formulation of primers to achieve the correct assembly of our constructs.
All of our primers needed to have a melting temperature (Tm) of around 60°C to maintain stability and anneal at
similar temperature during our PCR reactions. We also aimed to prevent the formation of hairpins in their secondary structures.
To begin with, we designed our insert primers by incorporating 20 base pairs from the vector (specifically, the overlap region)
and 15 base pairs from our insert.
We used IDT’s oligoanalyzer tool to examine both the melting temperature (Tm) and the secondary structure of our primers.
If the analysis revealed the formation of more than 3-4 hydrogen bonds within the primer's nucleotides, it would indicate
an unfavorable secondary structure. In that case, we would need to make adjustments, such as changing certain bases or
incorporating extra bases in the primer region of our insert.
Additionally, we examined the potential for self-dimerization in our primers.
Here are some examples of our primer optimization process:
Forward primer hemL
5’ GCC AGC ACA TGG ACA GCC CAA TGA GTA AGT CTG AA 3’
Forward primer hemL before optimization - Secondary structure
Within this primer's secondary structure, six hydrogen bonds were observed. Since this conformation was not suitable for
an effective primer, we proceeded to numerous optimization attempts. After multiple trials, we concluded that the addition
of six extra nucleotides at the beginning of our insert and the redesign of our primer to still include 20bp of our vector
and 15bp of our inserts provided the best optimization for the primer's secondary structure.
Forward primer hemL after optimization:
5’GCC AGC ACA TGG ACA GCC CAG CGA AGA TGA GTA AG 3’
Forward primer hemL after optimization - Secondary structure
Forward primer hemD
5’ TAT CAA CAG GAG TCC AAG CGT AAG TCG AAC AGA AA 3’
Forward primer hemD before optimization - Secondary structure
Within this primer's secondary structure, six hydrogen bonds were observed. Since this conformation was not
suitable for an effective primer, we proceeded to numerous optimization attempts. After multiple trials,
we concluded that the addition of six extra nucleotides at the beginning of our insert and the redesign
of our primer to still include 20bp of our vector and 15bp of our inserts provided the best optimization
for the primer's secondary structure.
Forward primer hemD after optimization
5’ TAT CAA CAG GAG TCC AAG CGT AAA TCT AAC AGA AA 3’
Forward primer hemD after optimization - Secondary structure
Build
We ordered our primers from IDT in order to begin our construct assembly experiments.
Test
We used the ordered primers to perform a PCR reaction for our inserts, introducing the desired overlap regions to their ends.
We also employed them for a
colony PCR to verify if the assembly of our constructs was successful.
Learn
Most of the primers we created worked well. This process educated us about the significance of optimizing
their structure and melting temperature (Tm), and it also gave us hands-on experience in achieving this optimization.
It is important to improve their structure and characteristics
before ordering them from a company to achieve better results.