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                         <p> Standard vectors for MoClo cloning are equipped with antibiotic resistance cassettes for cloning in <i>E. coli</i>. But since this work is focused on the expression of PET degrading enzymes by <i>C. reinhardtii</i>, the final transformable devices need to provide a selection advantage for these algae. Pierre Crozet and his team created a MoClo toolkit adapted for the model organism C. reinhardtii with 119 openly distributed genetic parts (Crozet et al., 2018) including resistance cassettes. However, these resistance cassettes are not located on any L1 plasmid backbone. Consequently, an additional cloning step is required to assemble a level 2 device, since a single level 1 module cannot be transformed without a resistance cassette.
 
                         <p> Standard vectors for MoClo cloning are equipped with antibiotic resistance cassettes for cloning in <i>E. coli</i>. But since this work is focused on the expression of PET degrading enzymes by <i>C. reinhardtii</i>, the final transformable devices need to provide a selection advantage for these algae. Pierre Crozet and his team created a MoClo toolkit adapted for the model organism C. reinhardtii with 119 openly distributed genetic parts (Crozet et al., 2018) including resistance cassettes. However, these resistance cassettes are not located on any L1 plasmid backbone. Consequently, an additional cloning step is required to assemble a level 2 device, since a single level 1 module cannot be transformed without a resistance cassette.
 
To simplify this cloning process, we design a <i>C. reinhardtii</i> specific resistance cassette to serve as a plasmid component for the transformation into <i>Chlamydomonas</i>.
 
To simplify this cloning process, we design a <i>C. reinhardtii</i> specific resistance cassette to serve as a plasmid component for the transformation into <i>Chlamydomonas</i>.

Revision as of 21:13, 2 October 2019

plasmid

Design

As previously explained on our Description page it is our aim to establish C. reinhardtii in the iGEM competition. To reach this goal we created a tool kit of various functional parts and multi-use constructs that future iGEM teams can use and optimize.

So, what is our focus?

1. Establishing C. reinhardtii as a platform in the competition

2. Working on the PET-degradation as a proof of concept

3. Building a bioreactor, in which we can cultivate C. reinhardtii and test its growth rates under different conditions

1.1 Golden Gate Modular Cloning

for Chlamydomonas reinhardtii

To synthesize and assemble the desired genetic elements, we applied the Type IIS “Golden Gate” cloning strategy (Engler et al., 2008). We used the Modular Cloning (MoClo) toolkit optimized for C. reinhardtii (Crozet et al., 2018), which follows the MoClo syntax of the plant synthetic biology community (Patron et al., 2015). Type IIS restriction enzymes cut outside their recognition sites, making them useful in this cloning method for consecutive assembly of fragments. Through the restriction, overhangs are formed which allow the fusion of said genetic fragments to complementary overhangs of the syntax and thereby determine the order of each in a transcriptional unit (Figure 1). These fusion sites allow for the assembly of several fragments in the right order in just one cloning reaction. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ functions.


Overview of the hierarchical and modular cloning system

Fig. 1. Universal MoClo fusion sites.

12 fusion sites (Patron, 2015) for the seamless fusion of different level 0 parts. In general, the fusion sites are grouped into 5’ untranslated regions including promoter sequences (grey), the translated coding sequence CDS (blue) followed by 3’ UTRs ending with a terminator (orange). Within these types, various parts (e.g. three different coding sequences) can be designed and combined into one transcription unit when cloned together into a L1 vector. The bold ATG within the B3 fusion site sets the transcription start.

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. Construction of an L0-part is done by flanking a gene of interest with the specific fusion site and the recognition site of BpiI by a PCR reaction. Upon digestion by BpiI it can be inserted into a previously digested L0-backbone. To then clone it into a L1-backbone, it is digested by BsaI, revealing the fusion sites for its assembly in a transcription unit. Lastly, a fusion of several transcription units (L1) into a L2 multigenic device is possible with the MoClo syntax.

As part of our contribution to a toolkit usable by future iGEM teams we design and construct not only the parts we intend to use on our goal of PET-degradation but several more, a L0-backbone and L1-backbone.

To ensure that all parts were designed correctly we cloned the PCR fragments into a L0 vector. To this end, we used a self-modified version of a L0 backbone containing RFP. After ligation, we transformed the L0 plasmids containing the parts into Escherichia coli and checked for white transformants. Only genes with the correct fusion and restriction sites could be inserted into the L0 backbone resulting in growth of white colonies, since the RFP gene was interrupted. For further verification, we checked the parts by DNA sequencing. We used the same control mechanisms for L1 assembly constructs.

cloning strategy

Fig. 2. Overview of the Golden Gate cloning strategy.

Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.

1.2 Construction of a selection cassette

Standard vectors for MoClo cloning are equipped with antibiotic resistance cassettes for cloning in E. coli. But since this work is focused on the expression of PET degrading enzymes by C. reinhardtii, the final transformable devices need to provide a selection advantage for these algae. Pierre Crozet and his team created a MoClo toolkit adapted for the model organism C. reinhardtii with 119 openly distributed genetic parts (Crozet et al., 2018) including resistance cassettes. However, these resistance cassettes are not located on any L1 plasmid backbone. Consequently, an additional cloning step is required to assemble a level 2 device, since a single level 1 module cannot be transformed without a resistance cassette. To simplify this cloning process, we design a C. reinhardtii specific resistance cassette to serve as a plasmid component for the transformation into Chlamydomonas.


To build a selection cassette using the Golden Gate cloning standard, each part required for a selection cassette (promoter, linker, marker and terminator) must be cloned into a level 0 backbone. The “marker” can be any antibiotic resistance for C. reinhardtii. Golden Gate cloning using BsaI can assemble the parts into a level 1 backbone which results in an antibiotic resistance transcriptional unit. Using primers amplifying the selection cassette on a L1 MoClo backbone attaching the restriction sites compared to restriction sites on the “new” L1a,b,c vectors. By attaching BamHI and XhoI sites a PCR product arises which is linkable with the new constructed L1 plasmids when digested with BamHI and XhoI. The resulting plasmids can then represent level 1 Golden Gate cloning vectors specified for C. reinhardtii. The picture shows an example to produce a selection cassette containing a hygromycin resistance linkable to a new L1c plasmid.

Selection cassette

Fig. 3. Cloning process to insert a selection cassette into the L1c-RFP_ampR/Ori plasmid.

The selection transcription unit (PsaD-B2Linker-hygR-Rbcs2) inside the pICH47732 backbone can be amplified with primers containing restriction sites for BamHI and XhoI to receive a PCR product linkable with the new constructed L1 plasmid L1c-RFP_ampR/Ori when digested with BamHI and XhoI. The resulting plasmid L1c-RFP-HygR represents a level 1 Golden Gate cloning vector specified for C. reinhardtii.

3. Designing and building a bioreactor

Usage of a Bioreactor