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

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             <h2 class="page-subheadline">Stuff that worked</h2>
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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>
 
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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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 for red-/white-selection. 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.  
 
                           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. 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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                        <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">Fluorescence of YFP</h3>
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                            The yellow fluorescent protein YFP was used as a fluorescent tag in some of our constructs. The goal of using YFP as a tag was the measurement of enzyme expression and secretion and to screen for successful mutants using YFP as a marker. Additionally, we wanted to use a YFP-expressing <i>C. reinhardtii</i> to analyse possible locus effects on expression. We were able to successfully transform a YFP-expressing <i>C. reinhardtii</i> with a construct of our own design (Part <a href="http://parts.igem.org/Part:BBa_K2984019">BBa_K2984019</a>).</br></br>
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We were able to successfully measure YFP fluorescence intensity and fluorescence spectra of YFP-expressing <i>C. reinhardtii</i> clones in comparison to the wild type (WT), although along the way we faced problems due to the autofluorescence by photosynthetic compartments. The results showed that our clone exhibited a higher fluorescence intensity at 528 nm than the WT (YFP emission peak) and the fluorescence spectrum of YFP confirmed the presence of the yellow fluorescent protein. This YFP-expressing clone also allowed us to characterize the light induction of the PsaD promoter by doing a time-resolved measurement of the fluorescence intensity. During this measurement we exposed <i>C. reinhardtii</i> cultures which contained our YFP construct to different growth conditions. One was exposed only to the dark, the other to synchronized growth conditions (10 hours dark, 14 hours illuminated). </br></br>
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Then, we started a time-resolved fluorescence intensity measurement in the dark, with a WT control. After approximately four hours, we activated a light source and exposed the cultures to light, thus activating the light inducible PsaD promoter. Our results showed that for the dark and synchronized cultures containing the YFP construct a peak in fluorescence intensity could clearly be seen after the light induction. This proved the light induction of the PsaD promoter. If you are interested in this measurement, please visit the page of our YFP mVenus construct in the iGEM registry <a href="http://parts.igem.org/Part:BBa_K2984019"> here</a>. </br></br>
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Revision as of 11:19, 20 October 2019

notebook

Demonstrate

Evidence for functionality of our system

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 for red-/white-selection. 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.
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.

Fluorescence of YFP

The yellow fluorescent protein YFP was used as a fluorescent tag in some of our constructs. The goal of using YFP as a tag was the measurement of enzyme expression and secretion and to screen for successful mutants using YFP as a marker. Additionally, we wanted to use a YFP-expressing C. reinhardtii to analyse possible locus effects on expression. We were able to successfully transform a YFP-expressing C. reinhardtii with a construct of our own design (Part BBa_K2984019).

We were able to successfully measure YFP fluorescence intensity and fluorescence spectra of YFP-expressing C. reinhardtii clones in comparison to the wild type (WT), although along the way we faced problems due to the autofluorescence by photosynthetic compartments. The results showed that our clone exhibited a higher fluorescence intensity at 528 nm than the WT (YFP emission peak) and the fluorescence spectrum of YFP confirmed the presence of the yellow fluorescent protein. This YFP-expressing clone also allowed us to characterize the light induction of the PsaD promoter by doing a time-resolved measurement of the fluorescence intensity. During this measurement we exposed C. reinhardtii cultures which contained our YFP construct to different growth conditions. One was exposed only to the dark, the other to synchronized growth conditions (10 hours dark, 14 hours illuminated). 

Then, we started a time-resolved fluorescence intensity measurement in the dark, with a WT control. After approximately four hours, we activated a light source and exposed the cultures to light, thus activating the light inducible PsaD promoter. Our results showed that for the dark and synchronized cultures containing the YFP construct a peak in fluorescence intensity could clearly be seen after the light induction. This proved the light induction of the PsaD promoter. If you are interested in this measurement, please visit the page of our YFP mVenus construct in the iGEM registry here.

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