Team:FCB-UANL/Results
Ligation
pSB3K3
Ligation
Rsn ASSEMBLY
RESULTS
The first thing in our itinerary was to carry out a bacterial transformation with E. coli Top 10 strain using the DNA vector pSB1C3 from the iGEM’s distribution kit plates. We would use this vector to clone our previously designed and synthesized DNA pieces. For this purpose, we used E. coli Top 10 Ca++ competent cells. In the next image we can observe the colonies of pSB1C3 in red, due to the presence of the RFP reporter gene.
Three of the transformed colonies were grown in 3 mL of LB medium containing chloramphenicol at 37°C. After overnight growth we carried out a plasmid DNA extraction of the pSB1C3 vector. To validate its identity, we made a 0.8% agarose gel electrophoresis. The results are shown in the following image, where the correct extraction of the plasmid DNA from the three overnight cultures can be observed.
Then, the obtained DNAs were digested with restriction enzymes using EcoRI and PstI enzymes in order to separate the RFP fragment from the plasmid, so that we could use it to assemble our DNA pieces in future steps. To ensure the correct DNA digestion and validate its identity, we made a 0.8% agarose gel electrophoresis. The results are shown in the following image, showing the release of the RFP fragment with a length of 1069 bp from the vector pSB1C3 with a length of 2070 bp. The length of every fragment was determined by comparison with the New England BioLabs Quick-Load® Purple 1 kb Plus DNA Ladder.
The following step was to carry out the ligation of the fragments corresponding to the Ranspumins DNA pieces that we received from Twist (Rsn-2 BBa_K3498009 , Rsn-3 BBa_K3498010 , and Rsn-4-5 BBa_K3498011 and BBa_K3498012 ) with the digested plasmid pSB1C3. For further information of the designed parts, visit our project description section . The ranaspumin pieces were previously digested using EcoRI and PstI enzymes to generate the sticky ends needed for their cloning in the plasmid. In order to test if the cloning process was successful, we carried out an E. coli Top 10 bacterial transformation using the ligated Ranaspumins (Rsn-2, Rsn-3, and Rsn-4-5) and pSB1C3 DNAs, where we observed bacterial growth in petri dishes containing LB agar and chloramphenicol. The next image shows in white the colonies that were selected for validation.
Next, three of the transformed colonies with Rsn-2, as well as three of the transformed colonies with Rsn-3 were grown in 3 mL of LB medium containing chloramphenicol at 37°C. After overnight growth, we carried out a plasmid DNA extraction from the culture and made a 0.8% agarose gel electrophoresis to validate its identity, which is shown in the next image.Rsn-4-5 was storage for its future usage.
Rsn-2 and Rsn-3 DNA fragments were digested with restriction enzymes using EcoRI and PstI, and we made a 0.8% agarose gel electrophoresis to validate its identity. The results are shown in the next image, where it can be observed pSB1C3 (2070 bp), Rsn-2 (1477 bp), and Rsn-3 (1591 bp) compared to the New England BioLabs Quick-Load® Purple 1 kb Plus DNA Ladder.
At this point, we already had our Ranaspumins pieces inserted in an E. coli Top10 strain in a high number of copies of plasmid, so we started working with the expression of the protein, detailedly explained in our engineering success section .
After the first production cycle, we decided to subclone Rsn-2 and Rsn-3 in the plasmid of a low number of copies, pSB3K3 (BBa_I20260 ). For this the vector was extracted from the iGEM distribution kit plates and E. coli Top 10 were transformed using the plasmid DNA. In the next image the colonies selected for pSB3K3 plasmid in fluorescent green, due to a GFP reporter gene can be observed.
After the first production cycle, we decided to subclone Rsn-2 and Rsn-3 in the plasmid of a low number of copies, pSB3K3 (BBa_I20260 ). For this the vector was extracted from the iGEM distribution kit plates and E. coli Top 10 were transformed using the plasmid DNA. In the next image the colonies selected for pSB3K3 plasmid in fluorescent green, due to a GFP reporter gene can be observed.
Three of the transformed colonies were grown in 3 mL of LB medium containing kanamycin at 37°C. After overnight growth we carried out a plasmid DNA extraction of the pSB3K3 vector. To validate its identity, we made a 0.8% agarose gel electrophoresis. The results are shown in the following image, where it can be observed the correct extraction of the plasmid DNA form the three overnight cultures
Then, the obtained DNAs were digested with restriction enzymes using EcoRI and PstI enzymes in order to separate the GFP fragment from the plasmid, so that we could use it to assemble our DNA pieces in future steps. To ensure the correct DNA digestion and validate its identity, we made a 0.8% agarose gel electrophoresis. The results are shown in the following image, observing the liberation of the GFP fragment with a length of 919 bp from the vector pSB3K3 with a length of 2750 bp. The length of every fragment was determined by comparison with the New England BioLabs Quick-Load® Purple 1 kb Plus DNA Ladder.
Using a Thermo Scientific™ NanoDrop 2000 we determined the concentration of both digested DNAs pSB3K3 and the ranaspumin pieces.
Then we carried out a ligation protocol to incorporate the digested Rsn-2 and Rsn-3 DNA to the pSB3K3 vector. Right after, we made a bacterial transformation to incorporate these DNA into E. coli Top 10. In the next image, the white colonies selected for validation can be observed.
Then we carried out a ligation protocol to incorporate the digested Rsn-2 and Rsn-3 DNA to the pSB3K3 vector. Right after, we made a bacterial transformation to incorporate these DNA into E. coli Top 10. In the next image, the white colonies selected for validation can be observed.
Once again, we carried out a plasmid DNA extraction from a 3 mL LB media with kanamycin overnight culture from three colonies previously selected for each ranaspumin, and made a 0.8% agarose gel electrophoresis to validate its identity.
In addition, we made a PCR amplification of the fragments and made another 0.8% agarose gel electrophoresis to ensure the identity. in the following image it can be observed the amplified fragments of Rsn-2 and Rsn-3 DNA in pSB3K3 vector, in comparison with Rsn-2 in pSB1C3 extracted plasmid DNA, Rsn-2 digested DNA, Rsn-3 digested DNA and Rsn-3 in pSB1C3 extracted plasmid DNA.
The next step was to assemble Rsn-3 and Rsn 4-5 together to have all the proteins produced on a single bacterial strain. For this, two colonies of the storaged Ranspumin 4-5 in pSB13 vector were grown in 3 mL media LB with chloramphenicol at 37°C overnight. From this culture the plasmid DNA was extracted as shown in the next image.
Then we digested both Rsn-3 and Rsn-4-5 DNA fragments with restriction enzymes: for Rsn-3 we used EcoRI and SpeI enzymes, while for Rsn 4-5 we used XbaI and PstI enzymes. Afterwards, we made a 0.8% agarose gel electrophoresis to validate its identity. It can be observed a single band for the linearized Rsn-3 and pSB3K3 DNA (4,341 bp) and the liberation of the Rsn-4-5 fragment (1424 bp) from pSB1C3 (2070 bp) in comparison with the New England BioLabs Quick-Load® Purple 1 kb Plus DNA Ladder.
After that, we used the DNA to make an E. coli top 10 bacterial transformation, in order to have our vector and pieces into E. coli. For validation, we made another DNA extraction from three previously grown colonies in 3 mL media LB with kanamycin at 37°C overnight followed by a 0.8% agarose gel electrophoresis for the extracted DNA. In the following image it can be observed Rsn-3-5 in pSB3K3 plasmidic DNA in comparison with Rsn-3 in pSB3K3 plasmid DNA and Rsn-4-5 in pSB1C3 plasmid DNA.
In the same way as previously done, we made a PCR amplification of the fragments and made another 0.8% agarose gel electrophoresis to ensure the identity. In the image it can be observed the Rsn-3-5 amplified fragment in comparison with the plasmid DNA extraction from Rsn-3-5, the linearized Rsn-3 and pSB3K3 DNAs and the digested Rsn-4-5 DNA.
Finally, we sequenced all the Ranaspumins fragments in a the PSB3K3 vector. We would like to thank the Biotechnology Institute of the UNAM for their help with the validation.
Our 2020-2021 iGEM project is generously supported by
