Week 14 (4/8-4/13): Working on scientific poster and passaging transformed cells

 Introduction

In lab this week, we did not perform any experiments because we mainly focused on editing and writing our scientific poster about our transformation project with E.coli plasmids and D. caeni for our conference on 4/13. 


The Poster

In the poster, we talked about the purpose of this experiment and we discussed about what Deinococcus species was successfully transformed in the past that allowed us to continuing working with E.coli plasmids and the Deinococcus species. In previous experiments, Deinoccocus radiodurans was proven to be successfully transformed with pRAD1. Below shows the results of the transformed cells on TGY plates with 3 ug/ml chloramphenicol. 

These plates show 1 ug of pRAD1. 

Below is another result of the transformed cells in a gel performed from colony PCR as confirmation. 


Lane 1: 100 BP Ladder

Lane 3-5: 861 BP of PCR product






Since we have confirmation and evidence that a Deinococcus species have the ability to be transformed with an E.coli plasmid, we wanted to expand this research with D. caeni as it was a primarily "unknown" species for the Deinococcus family and have not yet been further researched. We decided to transform D. caeni with pRAD1 and pwtCas9. 

In the poster, we rendered an illustration of our transformation procedure. 

  

NOTE: Our D. caeni and pRAD1 was grown on TGY agar with 3.5 ug/ml of chloramphenicol while our D. caeni and pwtCas9 was grown on TGY agar with 2 ug/ml of tetracycline. 

Below shows the plate results of our transformed D. caeni and pRAD1 cells and the plasmid prep gel. 

GEL:  Lane 1-2: pRAD1 plasmid extraction from D. caeni     Lane 3: 1 KP ladder     

Lane 4: pRAD 1 plasmid extraction from E.coli 

  


Below shows the plate results of our transformed D. caeni and pwtCas9 cells and plasmid extraction gel. 

GEL: Lane 1: 1 KP Ladder     Lane 4-6: pwtCas9 plasmid extraction from E.coli 


Discussion

In our discussion section, we discussed about the potential future for this project and what possibility this research can open up for other Deinococcus species and their relations with E.coli and E.coli plasmids. We plan to work more on the D. caeni and pwtCas9 cells as we involve the co-introduction into D. caeni of plasmids carrying guide RNA for the purposes of Cas9 mediated mutagenesis.

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