Difference between revisions of "Part:BBa K5078004"

 
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=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  
 
this part includes the psad promoter with 5' utr and the psad terminator to allow for spectinomycin resistance to be expressed in Chlamydomonas reinhardtii  
  
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===Usage and Biology===
 
===Usage and Biology===
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Our pL1-aadA is a level 1 construct containing the Psad Promoter+5' UTR (BBa_K3002001), the basic part aadA CDS that encodes for spectinomycin resistance for Chlamydomonas reinhardtii (BBa_K3002000), and the Psad Terminator (BBa_K3002002). pL1-aadA is used in our fully completed level 2 plasmids (BBa_K5078009 and BBa_K5078010) to give Chlamydomonas reinhardtii spectinomycin resistance. Which act as one of our selection markers to determine that our plasmid was completely integrated into C. reinhardtii.
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<img src="https://static.igem.wiki/teams/5078/plasmid-pictures/pl1-aada.webp" width="400" height="auto"/><br>Figure 1. Plasmid diagram of pL1-aadA using benchling for modeling.
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===Plasmid Verification===
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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.
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<img src="https://static.igem.wiki/teams/5078/electroporation.webp" width="400" height="auto"/><br>Figure 2. 100 uL of the electroporated C. reinhardtii with linerized pL1-aadA on Spectinomycin and TAP plate after two weeks of growth.
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Revision as of 19:46, 28 September 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 that encodes for spectinomycin resistance for Chlamydomonas reinhardtii (BBa_K3002000), and the Psad Terminator (BBa_K3002002). pL1-aadA is used in our fully completed level 2 plasmids (BBa_K5078009 and BBa_K5078010) to give Chlamydomonas reinhardtii spectinomycin resistance. Which act 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1401
    Illegal NgoMIV site found at 1584
    Illegal NgoMIV site found at 1694
  • 1000
    COMPATIBLE WITH RFC[1000]