Biology
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Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit
Chapters
Summary October 31st, 2019
Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.
Transcript
Cyanobacteria are an important phylum of photosynthetic microbes that are increasingly recognized as useful platforms for synthetic biology and renewable biotechnology applications. So developing robust techniques to simplify the design, cloning, and introduction of heterologous DNA into different cyanobacterial species is important. So this technique demonstrates a well-established method called conjugation or triparental mating for introducing DNA into cyanobacterial species that are not naturally transformable.
Conjugation has been successfully performed in a wide array of cyanobacterial strains from single cell to multicellular filamentous species. If you are attempting this technique for the first time, please take care to handle both your cyanobacterial and your E.coli strains with care. For example, do not centrifuge above the values given in the protocol and do not vortex.
Mixing bacteria and Cyanobacteria may seem strange to first-time users. We hope that by demonstrating the conjugation process, we will help to clarify this. After construction of level one assemblies, choose an appropriate level T acceptor vector.
Choose an appropriate end link to ligate the three prime end of the final level one assembly to the level T backbone. To assemble one or more level one assemblies in level T, prepare a reaction mix with PBI1 and the required end link vector. Set up the thermal cycler program and proceed according to the manuscript.
On day one, grow cyanobacterial culture by setting up a fresh culture of Synechocystis PCC 6803 or Synechococcus elongatus UTEX 2973. With a heat sterile loop, scrape the cells from the axenic BG-11 agar plate and place into a 100 milliliter conical flask filled with 50 milliliters of fresh BG-11 medium to inoculate. Place the flask into a shaking incubator with illumination to grow the Synechocystis PCC 6803 cell cultures at 30 degrees Celsius at 100 RPM.
For Synechococcus elongatus UTEX 2973, grow the cell cultures at 40 degrees Celsius at 100 RPM. After one to two days, draw one milliliter of the cell culture and measure on a spectrophotometer. The absorbance at OD 750 reaches 0.5 to 1.5.
On day two, inoculate an MC1061 E.coli helper strain containing vectors PRK24 and PRL528 in five milliliters of LB medium with ampicillin at a concentration of 100 micrograms per milliliter and chloramphenicol at a concentration of 25 micrograms per milliliter. Grow at 37 degrees Celsius overnight at 225 RPM in a shaking incubator. Then inoculate five milliliters of LB medium containing appropriate antibiotics with the E.coli culture carrying the level T cargo vector.
Grow the culture at 37 degrees Celsius overnight at 225 RPM in a shaking incubator. On day three, centrifuge the helper and the cargo E.coli overnight cultures at 3, 000 g for 10 minutes at room temperature. Discard the supernatant without disturbing the cell pellet.
Wash the pellets by adding five milliliters of fresh LB medium without antibiotics. Resuspend the pellet by gently pipetting up and down and centrifuge to remove the supernatant. Repeat this washing step three times to remove residual antibiotics from the overnight culture.
After the last wash, resuspend the cell pellet in half the volume of LB medium of the initial culture volume. Then combine 450 microliters of the helper strain with 450 microliters of the cargo strain in a two milliliter tube and leave at room temperature. Next, prepare the cyanobacterial culture by centrifuging one milliliter of cyanobacterial culture at 1, 500 g for 10 minutes at room temperature.
Then discard the supernatant carefully without disturbing the cell pellet. Wash the pellet four times by adding fresh BG-11 medium of the same initial volume. Add 900 microliters of the washed cyanobacterial culture to 900 microliters of the combined E.coli strains in a two milliliter tube.
Mix the cultures by gently pipetting up and down. Incubate the mixture at room temperature for 30 minutes for Synechocystis PCC 6803 or two hours for Synechococcus elongatus UTEX 2973. Centrifuge the mixture at 1, 500 g for 10 minutes at room temperature.
Remove 1.6 milliliters of the supernatant and resuspend the pellet in the remaining supernatant. Place one 0.45 micron membrane filter on an LB BG-11 agar plate without antibiotics. Carefully spread 200 microliters of the E.coli/cyanobacterial culture mix on the membrane with a sterile spreader or a sterile bended tip.
Seal the plate with paraffin film and place the plate into an illuminated incubator for 24 hours. After 24 hours, carefully transfer the membrane using flame sterilized forceps to a fresh BG-11 agar plate containing appropriate antibiotics to select for the cargo vector. Seal the plate with paraffin film and incubate under the same conditions as previously until colonies appear.
Colonies typically appear after 7-14 days for Synechocystis PCC 6803 and 3-7 days for Synechococcus elongatus UTEX 2973. To select conjugants, using a heat sterile inoculating rod, select at least two individual colonies from the membrane and streak onto a new BG-11 agar plate containing appropriate antibiotics. Then inoculate a streak of cyanobacterial culture into a 15 milliliter centrifuge tube containing five milliliters of LB medium and incubate at 37 degrees Celsius overnight at 225 RPM in a shaking incubator.
The clear culture confirms the absence of E.coli contamination. Following a sufficient growth period of seven days, pick individual axenic colonies to set up liquid cultures for long-term cryo storage or subsequent experimentation. In this study, golden gate assembly workflow was demonstrated.
Following transformation of the assembly reaction, successful assemblies were identified using standard blue white screening of E.coli colonies. The eYFP expression cassette in the level one vector and the end link one vector were then assembled into a level T acceptor vector to give the vector cpcBA-eYFP. The assembled cpcBA-eYFP vector was verified by restriction digestion.
Successful conjugal transfer of cpcBA-eYFP or the pPMQAK1-T vector resulted in the growth of up to several hundred colonies on the membrane for Synechocystis PCC 6803 and Synechococcus elongatus UTEX 2973. As expected for the strong Pcpc560 promoter, the values for normalized eYFP fluorescence from the plate reader and eYFP fluorescence per cell from the flow cytometer were high compared to the negative control. With both the plate reader and the flow cytometer, fluorescence values were higher in Synechococcus elongatus UTEX 2973 than in Synechocystis PCC 6803.
Ensure you incubate for the minimum time according to which strain you are conjugating.
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