Team:TU Kaiserslautern/Composite
Parts
In this page we collected all assembled constructs, both Level 1 and Level 2. Level 1 are a functional transcriptional unit, while level 2 constructs can contain multigene constructs. Since we were unable to test all of our Level 2 constructs, only the ones tested and registered will be listed.
Composite Parts for C. reinhardtii
Our composite parts for the green algae Chlamydomonas reinhardtii have been assembled with the Modular Cloning System, which is based on Golden Gate Assembly. It is founded on the use of Type IIS restriction enzymes, which cut outside their recognition site, and T4-DNA-ligase which allows a simultaneous assembly of several DNA-fragments in the intended order.1 You can see our composite parts listed below.
Table 1: All tested Level 1 and Level 2 constructs.
| Name | Type | Description | Designer | Length [bp] |
|---|---|---|---|---|
| BBa_K3589201 | Composite | L1 - Mutant BaLac + HA | Yannik Schermer | 4020 |
| BBa_K3589202 | Composite | L1 - Wildtype marLac + HA | Yannik Schermer | 3275 |
| BBa_K3589203 | Composite | L1 - Mutant BaLac + cCA + SP20-HA-RGS-8His | Yannik Schermer | 4206 |
| BBa_K3589204 | Composite | L1 - Wildtype marLac + cCA + SP20-HA-RGS-8His | Yannik Schermer | 3445 |
| BBa_K3589205 | Composite | L1 - Mutant BaLac + cCA + HA-RGS-8His | Yannik Schermer | 4080 |
| BBa_K3589206 | Composite | L1 - Wildtype marLac + cCA + HA-RGS-8His | Yannik Schermer | 3319 |
| BBa_K3589207 | Composite | L2 - BaLac_HA + SpecR | Yannik Schermer | 6436 |
| BBa_K3589208 | Composite | L2 - marLac_HA + SpecR | Yannik Schermer | 5701 |
| BBa_K3589209 | Composite | L2 - BaLac_cCA_SP20-HA-RGS-8His + SpecR | Yannik Schermer | 6622 |
| BBa_K3589210 | Composite | L2 - marLac_cCA_SP20-HA-RGS-8His + SpecR | Yannik Schermer | 5861 |
| BBa_K3589211 | Composite | L2 - BaLac_cCA_HA-RGS-8His + SpecR | Yannik Schermer | 6496 |
| BBa_K3589212 | Composite | L2 - marLac_cCA_HA-RGS-8His + SpecR | Yannik Schermer | 5735 |
Composite Parts – Level 1
Level 1 parts are made of at least a promotor, a coding sequence and a terminator; thus, forming a functioning eukaryotic transcriptional unit. Additionally, secretion signals and tags for protein detection can and have been added. Level 1 constructs have an ampicillin resistance for selection. 1 Table 2: All tested Level 1 constructs.
| Name | Type | Description | Designer | Length [bp] |
|---|---|---|---|---|
| BBa_K3589201 | Composite | L1 - Mutant BaLac + HA | Yannik Schermer | 4020 |
| BBa_K3589202 | Composite | L1 - Wildtype marLac + HA | Yannik Schermer | 3275 |
| BBa_K3589203 | Composite | L1 - Mutant BaLac + cCA + SP20-HA-RGS-8His | Yannik Schermer | 4206 |
| BBa_K3589204 | Composite | L1 - Wildtype marLac + cCA + SP20-HA-RGS-8His | Yannik Schermer | 3445 |
| BBa_K3589205 | Composite | L1 - Mutant BaLac + cCA + HA-RGS-8His | Yannik Schermer | 4080 |
| BBa_K3589206 | Composite | L1 - Wildtype marLac + cCA + HA-RGS-8His | Yannik Schermer | 3319 |
Composite Parts – Level 2
Fig 1: Overview of all designed Level 2 constructs for Chlamydomonas reinhardtii. All were transformed in C. reinhardtii. The ones with the green background were screened.
Several Level 1 constructs can be cloned into a Level 2 destination vector, which permits the construction of a multigene construct. This procedure is a quick alternative compared to co-transformation and allows a multigene construct consisting of up to 8 Level 1 modules. Since all 8 positions are seldomly needed, an end-linker has to be used to connect the used Level 1 parts to the Level 2 destination vector.1
Table 3: All tested Level 2 constructs.
| Name | Type | Description | Designer | Length [bp] |
|---|---|---|---|---|
| BBa_K3589207 | Composite | L2 - BaLac_HA + SpecR | Yannik Schermer | 6436 |
| BBa_K3589208 | Composite | L2 - marLac_HA + SpecR | Yannik Schermer | 5701 |
| BBa_K3589209 | Composite | L2 - BaLac_cCA_SP20-HA-RGS-8His + SpecR | Yannik Schermer | 6622 |
| BBa_K3589210 | Composite | L2 - marLac_cCA_SP20-HA-RGS-8His + SpecR | Yannik Schermer | 5861 |
| BBa_K3589211 | Composite | L2 - BaLac_cCA_HA-RGS-8His + SpecR | Yannik Schermer | 6496 |
| BBa_K3589212 | Composite | L2 - marLac_cCA_HA-RGS-8His + SpecR | Yannik Schermer | 5735 |
References
(1) Weber, E.; Engler, C.; Gruetzner, R.; Werner, S.; Marillonnet, S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 2011, 6 (2), e16765. DOI: 10.1371/journal.pone.0016765.
