Gene drive

Design Assignment

Name a Gene Drive:

1. What is the purpose of Daisy Quorum gene drive?

The technology can restore any drive-amenable population carrying engineered genes to wild-type genetics. Daisy quorum systems may enable efficient,community-supported, and genetically reversible ecological engineering.

2. What is your immediate emotional response after reading the preprint?

After reading the preprint, I was so carried away with the technology, that's so powerful in genetic engineering. I am still not able to believe how such things could be researched.

3. Describe a scenario, real or fictional(real world is preferred), where you would deploy a technology such as Daisy Quorum".

I would make India pest and insects such as ants and mosquitoes free using Daisy Quorum Technology, though a great deal of research for biological and social safety has to be considered before implementation.

4. What would you be concerned about if a neighboring country, or city, decided to deploy?

I would be concered about the safety of the society as the unintended damage to society will be very severe and long lasting.

5. Is the name “Daisy Quorum” intuitive to you? If you were to name the technology, what would you name it? Note: we may actually adopt your name in the final publication!“Daisy Quorum” technology?

No, the Daisy Quorum technology was not intuitive to me. I couldn't think of any other apt name for the technology.

Experimental Assignment

Make the following mixtures with the plasmids you were provided.Make sure that the total DNA concentration does not exceed 100ng/ul, and try to conserve materials. 10ul of plasmid mix is plenty for this experiment.

Transformation of E.Coli competent cells with the EC-Rope plasmid using heat- shock transformation method.

• The E.coli competent cells were kept on ice.
• We added around 3ul of the EC- Rope plasmid to the competent cells
• The cells were mixed by gently flicking the eppendorf.
• A water bath at 42 degrees was kept in a magnetic stirrer
• As soon as the EC- rope plasmid was added, the cells were kept in 42 degrees for 90 seconds and then at ice for 10 minutes.
• After the transformation, 500ul of LB media was added to the cells and kept in shaker at 37 degrees for 1 hour.
• The cells were then spinned down at 6000 rpm for 3.5 minutes.
• The cells were later plated on LB agar plates with 25ug/ml Carbenicillin and incubated at 37 degrees.
• The colonies were observed in the plate after incubation at 37 degrees overnight.



Preparation of new competent cells that contain the EC Rope plasmid

• The colonies grown overnight was inoculated in 5ml of LB medium with 25ug/ml overnight.
• Once the growth was observed, we centrifuged for 5 minutes at 6000 RPM and promptly removed the supernatant.
• 10ml of chilled glycerol was added to resuspend the pellet and centrifuged for 3.5 minutes at 6000 RPM.
• After washing with glycerol for 4 to 5 times, the cells were centrifuged for 3.5minutes at 6000 RPM.
• 100ul of 10% glycerol was added to the washed pellet.
• 2 aliquots of 50ul cells were sealed with parafilm and freezed until use.



Preparation of the plasmid mixtures

• 10ul of CRISPR-A mixture with Ds-cas9 + PM-bla and CRISPR-B mixture with Ds-cas9 + PM-sp22a-!con was prepared.




Transformation of competent cells with the two plasmid mixtures from step 4

• The 2 aliquots of competent cells were kept in ice and about 3ul of CRISPR-A and CRISPR-B was added to the 2 aliquots of competent cells.
• The cells after gently flicking were kept at 42 degrees for 90 seconds and then shifted to ice for 10 minutes.
• 500 ul of LB media was added to the transformed cells (both CRISPR-A and CRISPR-B) and kept in shaker at 37 degrees for 1 hour.
• The cells were then spinned down at 6000rpm for 3.5 minutes.



Plating the CRISPR-A and CRISPR-B cells

• The CRISPR-A and CRISPR-B cells were plated on double selective LB-Agar plates containing Spectinomycin at 50 ug/mL, and Chloramphenicol at 35 ug/mL
• The plates were incubated at 37 degrees overnight





Why did colonies carrying the CRISPR B mix grow and the CRISPR A mix fail? Hypothesize what happened. Feel free to use the plasmid maps provided as a guide.

The CRISPR-B has antibiotic resistance gene which caused the colonies to grow and the CRISPR-A did not have any resitance gene, so the growth of the bacteria was inhibited using the antibiotics.

New ethics or safety considerations this week:

Do your activities this week raise new ethics and/or safety considerations you had not considered in week 1? Describe what activities have raised these considerations and any changes you have implemented in response.

• I found that CRISPR technology can damage DNA that is far from target DNA to get it corrected. So enough caution was exercised during transformation and plating the cells.