Difference between revisions of "Team:Humboldt Berlin/Demonstrate"

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             <h2 class="page-subheadline">Stuff that worked</h2>
  
 
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                        <figcaption>Fig. 1 - Our self-constructed L0-RFP backbone transformed into <i>E. coli</i> (strain DH10B). Through red-/white-selection successful clones were picked and streaked out on LB-agar plates.</figcaption>
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                        <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/0/0d/T--Humboldt_Berlin--L0-RFP_AR.jpeg" alt="L0-RFP_AR" width="500" />
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                        <figcaption>Fig. 2 - AR promoter in L0-RFP-backbones expressed by <i>E. coli</i> (strain DH10B). The white clones represent positive transformants.</figcaption>
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                         <h3 class="headline3">Synthesized MoClo backbones</h3>
 
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                             To synthesize the genetic elements for the Chlamy-HUB Collection, we based our design on the Modular Cloning (MoClo) toolkit optimized for <i>C. reinhardtii</i> (Crozet et al., 2018), which follows the Golden Gate cloning method. To read more about the background on the cloning method please visit our <a href="https://2019.igem.org/Team:Humboldt_Berlin/Design#GoldenGate">Design page </a>.</br></br>
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Within the MoClo syntax, there are three different cloning vectors, level 0, 1 and 2 (referred to as "L0", "L1" and "L2", respectively). L0 vectors carry one basic genetic fragment or part, L1 vectors are assembled fragments creating a transcriptional unit and L2 are multigenic constructs. As part of our contribution to the iGEM Registry, the ChlamyHUB Collection, we registered two cloning vectors. The backbones to these vectors, L0-RFP- and L1-backbones were created by ourselves.</br></br>
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The L0-RFP (Part <a href="http://parts.igem.org/Part:BBa_K2984010">BBa_K2984010</a>) contains an origin for bacterial replication (Ori), a resistance gene against spectinomycin for expression by <i>E. coli</i> and the red fluorescent protein RFP. It is flanked by restriction sites for <i>Bpi</i>I. Parts with matching L0-overhangs according to the MoClo syntax can be inserted into the backbone using <i>Bpi</i>I. Successful transformants are white colored. The established backbone worked as intended, demonstrated by successful transformations of all L0-Parts we have designed. </br></br>
 
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                          Furthermore, we designed a L1-backbone (Part <a href="http://parts.igem.org/Part:BBa_K2984002">BBa_K2984002</a>) on which to assemble the transcriptional units for transformation into <i>C. reinhardtii</i>. It contains RFP, a resistance gene against ampicillin and an Ori for <i>E. coli</i> and is flanked by <i>Bsa</i>I restriction sites. L1-constructs can be transformed into and be expressed by <i>C. reinhardtii</i>. Thus it is proven, that the L1 backbone works properly.
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<figcaption>Fig. 3 - This PCR of <i>C. reinhardtii</i> clones that could grow on selective media shows bands at a length of 1,6 kb for successfully transformed clones. These were further verified by sequencing.</figcaption>  
 
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                         <p>Crozet, P., Navarro, F. J., Willmund, F., Mehrshahi, P., Bakowski, K., Lauersen, K. J., ... Lemaire, S. D. (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology, 7(9), 2074-2086. Retrieved from https://doi.org/10.1021/acssynbio.8b00251. doi:10.1021/acssynbio.8b00251
 
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Revision as of 10:52, 20 October 2019

notebook

Demonstrate

Stuff that worked

L0-RFP-clone
Fig. 1 - Our self-constructed L0-RFP backbone transformed into E. coli (strain DH10B). Through red-/white-selection successful clones were picked and streaked out on LB-agar plates.
L0-RFP_AR
Fig. 2 - AR promoter in L0-RFP-backbones expressed by E. coli (strain DH10B). The white clones represent positive transformants.

Synthesized MoClo backbones

To synthesize the genetic elements for the Chlamy-HUB Collection, we based our design on the Modular Cloning (MoClo) toolkit optimized for C. reinhardtii (Crozet et al., 2018), which follows the Golden Gate cloning method. To read more about the background on the cloning method please visit our Design page .

Within the MoClo syntax, there are three different cloning vectors, level 0, 1 and 2 (referred to as "L0", "L1" and "L2", respectively). L0 vectors carry one basic genetic fragment or part, L1 vectors are assembled fragments creating a transcriptional unit and L2 are multigenic constructs. As part of our contribution to the iGEM Registry, the ChlamyHUB Collection, we registered two cloning vectors. The backbones to these vectors, L0-RFP- and L1-backbones were created by ourselves.

The L0-RFP (Part BBa_K2984010) contains an origin for bacterial replication (Ori), a resistance gene against spectinomycin for expression by E. coli and the red fluorescent protein RFP. It is flanked by restriction sites for BpiI. Parts with matching L0-overhangs according to the MoClo syntax can be inserted into the backbone using BpiI. Successful transformants are white colored. The established backbone worked as intended, demonstrated by successful transformations of all L0-Parts we have designed.

Headline

Furthermore, we designed a L1-backbone (Part BBa_K2984002) on which to assemble the transcriptional units for transformation into C. reinhardtii. It contains RFP, a resistance gene against ampicillin and an Ori for E. coli and is flanked by BsaI restriction sites. L1-constructs can be transformed into and be expressed by C. reinhardtii. Thus it is proven, that the L1 backbone works properly.

L1c-construct1
Fig. 3 - This PCR of C. reinhardtii clones that could grow on selective media shows bands at a length of 1,6 kb for successfully transformed clones. These were further verified by sequencing.

Crozet, P., Navarro, F. J., Willmund, F., Mehrshahi, P., Bakowski, K., Lauersen, K. J., ... Lemaire, S. D. (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology, 7(9), 2074-2086. Retrieved from https://doi.org/10.1021/acssynbio.8b00251. doi:10.1021/acssynbio.8b00251