Experiments
In this page you will find the respective workflows of our different wet lab procedures.
Overview
During this cycle, our team performed a diversity of experiments to achieve our objectives. The iGEM-RUM cloning plan was divided into two devices, with Device 1: AHL and luxR generator using DNA from the iGEM DNA Kit Plates and Device 2: Biodegradation of RDX, using DNA designed by iGEM-RUM 2021 and subsequently ordered from Integrated DNA Technologies (IDT). Cloning for these parts into our EC100™ E. coli competent cells was done by following a simple restriction endonuclease digestion protocol for both devices in which they were digested using the prefixes and suffixes provided to us by iGEM. Digestion of our individual inserts was followed by ligation and transformation into our pSB1C3 vector. From here, subsequent ligation of both devices into one same cell was intended so that the cell could be functional for AHL and LuxR generation and RDX biodegradation.The final cloning plan for our Device 1 and Device 2 followed the workflow shown below with their respective restriction sites and restriction endonucleases as seen in Figures 1 and 2.
Previous Cloning Plan
Device 1
During our time working in the lab, our cloning plan was optimized twice, for a total of three cloning strategies. However, due to an unforeseen meteorological event which compromised our samples, we were unable to finalize the last cloning strategy. Our initial cloning strategy for Device 1 (the supplementary version) consisted of digesting iGEM’s linearized plasmid, pSB1A3, with EcoRI-HF and PstI-HF. D1P1 (BBa_K876014) was digested with EcoRI and SpeI while D1P2 (BBa_K081014) was digested with XbaI and PstI-HF. The resulting ligation would be digested with SpeI-HF and PstI-HF for the insertion of D1P3 (BBa_K116640) digested with XbaI and PstI-HF.
For our second cloning strategy, we utilized the digested pSB1C3 plasmid from the iGEM kit plate parts (D1P2 and D1P3), extracted and purified from agarose gel. Since we no longer used the pSB1A3 linearized backbone, we opted for a simpler insertion method. D1P1 would serve as the vector for D1P2 and D1P3 inserts. In order to linearize the plasmid while keeping the D1P1 insert, we digested D1P1 with SpeI-HF. Then, D1P2 was digested with XbaI and SpeI-HF producing a downstream insertion after ligation (A1). The A1 construct was then linearized with SpeI-HF for the insertion of D1P3. Like D1P2, D1P3 was digested with XbaI and SpeI-HF.
Device 2
Similar to Device 1, we employed the use of restriction cloning as our cloning strategy for Device 2. The genetic circuit for this device was designed in Benchling and divided into two fragments; both were ordered through Integrated DNA Technologies. These were delivered as linearized fragments which contain iGEM’s restriction site prefix and suffix. The cloning strategy for Device 2 changed twice. The initial cloning strategy for Device 2 consisted of using EcoRI-HF and SpeI-HF enzyme master mix alongside a XbaI and PstI-HF enzyme master mix for Device 2 Fragment 1 (D2F1) and Device 2 Fragment 2 (D2F2), respectively. Then, iGEM’s linearized vector, pSB1K3, was digested with a EcoRI-HF and PstI-HF enzyme master mix. After successful ligation, the result would be a circularized plasmid with D2F1 located upstream from D2F2.
Final Cloning Plan
Our final cloning plan for Device 1 consists of simple restriction endonuclease digestion. This device contains a pSB1C3 vector, the Plac-luxI composite (D1P1) BBa_K876014, the RFP protein generator (D1P2) BBa_K081014, and the LuxR pTet regulated (D1P3) BBa_K116640. To assemble Device 1 we digested D1P1 at the SpeI-HF and PstI-HF restriction sites and D1P2 with XbaI and PstI-HF. The idea behind selecting these sites is because for D1P1, we wanted to maintain the part and backbone attached (D1P1-BB). However, for D1P2 we needed to detach the part from its backbone. This site also ensured D1P2 was inserted downstream in D1P1-BB. After inactivating the digestion reactions, D1P1-BB and D1P2 were ligated for five hours in the thermocycler to secure a consistent temperature. The product of this ligation was named A1. Since the restriction sites (iGEM prefix and suffix) are conserved after ligation, the same steps performed for A1 were then repeated for A2: the insertion of the third part (D1P3) into the vector containing the first and second parts. This would complete our cloned Device 1.
Figure 1. Supplementary Device 1 Workflow
For Device 2, the final restriction endonuclease cloning plan was done by using our first fragment of the second device, D2F1, and ligating it to our vector backbone psB1C3. This backbone was extracted from D1P1 to ensure functionality of the vector. This first assembly was named B1. This procedure was done using the enzymes XbaI and SpeI-HF to digest our vector and insert D2F1. After this, a second digestion took place to insert the second fragment, D2F2, into B1. For this, we used the endonucleases SpeI-HF and PstI-HF to linearize B1 and used XbaI and PstI-HF to cut D2F2. Once we ligated our parts into a single vector the second assembly, B2, was generated culminating our Device 2.
Figure 2. Device 2 Workflow
Workflow
Both of our devices followed a similar cloning procedure. The traditional cloning by restriction endonuclease digest that was performed in the laboratory to insert our Device 1 into the EC100™ E. coli competent cells used the following general protocol:
Resuspension (Fig. 3) was conducted with the iGEM DNA Kit Plates retrieved from the iGEM Distribution Kits using the parts 22M and 6C by utilizing the corresponding wells to acquire the genes of interest following the protocol established by iGEM in 2019. The genes of interest were subsequently transformed into competent EC100™ E. coli cells by electroporation and left to grow overnight.
Figure 3. Workflow for Device 1
This was followed by restriction endonuclease digestion, which was performed utilizing type II restriction endonucleases EcoRI and XbaI prefixes and SpeI and PstI suffixes (Fig. 3) following the protocol from New England Biolabs.
To confirm these parts had been successfully cloned into the competent E. coli cells and digested correctly by the restriction endonucleases, gel extraction and purification was performed following the Monarch Gel Extraction Kit (Fig. 4) by excising DNA samples directly from the low melting point agarose gel, followed by purification of the gel to be ligated as one genetic circuit.
Figure 4. DNA Agarose Gel Extraction and Purification
After confirming all of the digested parts that were to be assembled eventually into a pSB1C3 vector, ligation was performed using the Cohesive End Ligation Protocol by New England Biolabs utilizing the Hi-T4 DNA Ligase (Fig. 1). Once ligation was performed, transformation of the competent cells was executed again with the assembled parts (Fig. 5) to complete the genetic circuit for Device 1 using the method mentioned previously for transformation of competent E. coli cells.
Figure 5.Transformation Protocol
The results from the transformation were plated into petri dishes (Fig. 3) prepared with the corresponding Chloramphenicol antibiotic as a selective measure and left overnight until colony growth was observed. Subsequently, the colonies that grew were inoculated into liquid medium using Luria Bertani Broth (Fig. 3) and left to grow overnight. This DNA underwent Qiagen Mini Prep to extract the plasmid DNA. Later on, this extracted DNA was put in gel electrophoresis to confirm that our DNA was successfully assembled.
The assembly of our second device, for biodegradation, followed a similar procedure (Fig. 6).
Figure 6. Workflow for Device 2
The general protocol differed in the primary steps of cloning, since our synthesized fragments for the Device 2 genetic circuit were ordered from Integrated DNA Technologies. Unlike the first device that came in a plasmid, the device 2 inserts came already linearized and thus needed to be digested before being ligated to a plasmid. Digestion of the inserts was done following the restriction endonuclease digestion procedure mentioned previously, according to their weight and calculations. This procedure was followed by agarose gel electrophoresis for confirmation of our individual parts and later on, gel extraction and purification to eventually ligate all of our individual parts into our plasmid of choice, pSB1C3. Once this procedure was done for both devices we would have proceeded to join both devices in a single chassis (Fig. 7).
Figure 7. Final Double Plasmid Insertion
References
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Protocol for Dephosphorylation of 5´-ends of DNA using CIP (NEB #M0290). (n.d.). New England Biolabs. https://international.neb.com/protocols/0001/01/01/protocol-for-dephosphorylating-with-cip
Protocol for Hi-T4TM DNA Ligase (NEB #M2622). (n.d.). New England Biolabs. https://international.neb.com/protocols/2019/10/10/protocol-for-hi-t4-dna-ligase-neb-m266
QIAGEN. (2020). Miniprep and Transformation Validation.
TA cloning method: Fragment Resuspension. (n.d.). Integrated DNA Technologies. https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/gblocks-fragment-t-a-cloning-protocol.pdf?sfvrsn=e41c3407_8
Thawing Frozen Stock Culture. (n.d.). ATCC. https://haveylab.horticulture.wisc.edu/wp-content/uploads/sites/66/2016/05/Preparation-of-frozen-bacterial-stock-cultures.pdf
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