Latest revision as of 00:20, 2 October 2024

pl1_aada for spectinomycin resistance in Chlamydomonas reinhardtii

this part includes the psad promoter with 5' utr and the psad terminator to allow for spectinomycin resistance to be expressed in Chlamydomonas reinhardtii

Usage and Biology

Our pL1-aadA is a level 1 construct containing the Psad Promoter+5' UTR (BBa_K3002001), the basic part aadA CDS, which encodes for spectinomycin resistance for Chlamydomonas reinhardtii (BBa_K3002000), and the Psad Terminator (BBa_K3002002). pL1-aadA is used in our fully completed De-Slimer plasmids (BBa_K5078009 and BBa_K5078010) to give Chlamydomonas reinhardtii spectinomycin resistance. This acts as one of our selection markers to determine that our plasmid was completely integrated into C. reinhardtii.

Figure 1. Plasmid diagram of pL1-aadA using benchling for modeling.

Plasmid Verification

To ensure that our pL1-aadA can be inserted and is expressed in C. reinhardtii we transform C. reinhardtii with just pL1-aadA. To do this we first linnerize the pL1-aadA by using the restriction enzyme BbsI. We do this because C. reinhardtii prefers liner DNA and we want to increase the likelihood of C. reinhardtii taking in pL1-aadA. After linearizing pL1-aadA we electroplate C. reinhardtii with 4ug of linerized DNA per 100uL of suspended cells. Following 24hrs of incubation, we centrifuge our sample. Following that we remove 3.8mL supernate and then add in 200uL of TAP media. Finally we add 100uL of the solution on spectinomycin and TAP plate, and then we incubate it for 5-7 days under a growth light.

Figure 2. 100 uL of the electroporated C. reinhardtii with linerized pL1-aadA on Spectinomycin and TAP plate after two weeks of growth.

Sequence and Features