Team:TU Kaiserslautern/Parts
Parts
On this page we present to you the parts we created with the MoClo system and worked with during the last few months. The MoClo system is based upon Golden Gate Assembly, which uses the unique characteristics of Type IIS restrictions enzymes and T4-DNA-ligase to create gene constructs.1 Additionally, we expressed the two laccases in the reliable model organism Escherichia coli. The two basic parts we created for E. coli were cloned in the pGEX-6P-1 expression vector and are also presented here.
For this year’s competition we decided to use two different chassis i.e. the green algae Chlamydomonas reinhardtii and the reliable model organism Escherichia coli. The E. coli experiments were carried out to gather large amount of proteins in a short period of time to characterize their activity.
Updating the part collection and sharing it allows us and future iGEM-teams to build upon these foundations. After all a continuous development is necessary to meet the demands of time.
All in all, we assembled 37 different constructs, with two laccases (the mutated BaLac, which is split into BaLac part 1 and BaLac part 2, and the wildtype marLac), three different secretion signals (cCA, GLE, ARS) and three different tags for detection (SP20-HA-8His-RGS, HA-8His-RGS, SP20-3HA). All constructs were transformed in C. reinhardtii but due to time restrictions, we were not able to screen all of them for expression of our protein of interest.
As seen in the above figure, Level 2 MoClo-constructs are assembled with an antibiotic resistance cassette for selection, followed by one of the secretion signals and the enzymes coding sequence. Due to time constraints, only the secretion signal cCA could be tested. Subsequently, a posttranslational modification tag was added.
If you are interested and want more information about our approach and the constructs themselves, please take a look at our part collection.
Table 1: Overview of all registered parts for C. reinhardtii
For Escherichia coli, both laccase coding sequences were placed into the pGEX-6P-1 expression vector. It was purchased from the company General Biosystems. This vector includes the tac-promotor, in combination with a lac-operator and a GST-tag, which has been added to our gene of interest, and a PreScission protease site tag. It also confers resistance to Ampicillin for an easy selection of positive transformants. See figure 2.
Table 2: Overview of all registered parts for E. coli
(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.
(2) Addgene Vector Database Plasmid: pGEX-6P-1. https://www.addgene.org/vector-database/2887/.
C. reinhardtii
For the experiments with C. reinhardtii, the MoClo system was used, same as our 2019's team used. By doing so, we built upon the Kaiser collection and using last year’s experiments could already pre-select the best working basic parts. Updating the part collection and sharing it allows us and future iGEM-teams to build upon these foundations. After all a continuous development is necessary to meet the demands of time.
All in all, we assembled 37 different constructs, with two laccases (the mutated BaLac, which is split into BaLac part 1 and BaLac part 2, and the wildtype marLac), three different secretion signals (cCA, GLE, ARS) and three different tags for detection (SP20-HA-8His-RGS, HA-8His-RGS, SP20-3HA). All constructs were transformed in C. reinhardtii but due to time restrictions, we were not able to screen all of them for expression of our protein of interest.
Fig. 1: Overview of composite parts or Level 2 constructs.
Table 1: Overview of all registered parts for C. reinhardtii
| Name | Type | Description | Designer | Length [bp] |
|---|---|---|---|---|
| BBa_K3589107 | Coding | Mutant BaLac for C. reinhardtii part 1 | Yannik Schermer | 1439 |
| BBa_K3589108 | Intermediate/Coding | Mutant BaLac for C. reinhardtii | Yannik Schermer | 2729 |
| BBa_K3589109 | Coding | Wildtype marLac for C. reinhardtii | Yannik Schermer | 1968 |
| BBa_K3589110 | Coding | Mutant BaLac for C. reinhardtii part 2 | Yannik Schermer | 1290 |
| BBa_K3589150 | Tag | SP20-HA-RGS-8His | Yannik Schermer | 195 |
| BBa_K3589151 | Tag | HA-RGS-8His | Yannik Schermer | 69 |
| 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 |
E. coli
Fig. 2: This figure shows the generated plasmid map of the expression vector pGEX-6P-1 with its restriction enzyme interface.2
| Name | Type | Description | Designer | Length [bp] |
|---|---|---|---|---|
| BBa_K3589105 | Coding | Mutant BaLac for E. coli | Yannik Schermer | 1686 |
| BBa_K3589106 | Coding | Mutant marLac for E. coli | Yannik Schermer | 1323 |
References
(2) Addgene Vector Database Plasmid: pGEX-6P-1. https://www.addgene.org/vector-database/2887/.
