Team:Amsterdam/test/joeri/index

Methods

Some methods are at the basis of our project and deserve some extra attention. Here, we will explain to you how you can make genetic modifications without introducing an antibiotics resistance cassette. We elaborate on our Multi-Cultivators, which we used to study the growth characteristics and phenotype of newly designed strains. Finally, we introduce you to our BioBrick TA-cloning system that was used throughout the entire project.

methods

Markerless genetic modification

Introduction

Traditionally, the introduction of an antibiotic resistance marker accompanies gene insertions or deletions such as the ones that we planned to implement in our project. However, input collected from several sources during our human practices activities, pointed out that it would be much better to avoid altogether the presence of resistance markers in the final production strains. Main reasons being that: (i) this makes the handling of the strains in industrial setting cheaper and easier; (ii) marker-free strains make further genetic modification independent of the availability of additional resistance markers; and (iii) the acceptance of our technology, given its added-biosafety, is increased as our newly designed strains will never be at risk of passing on to natural environments resistance cassettes.
Our team has decided to adopt the markerless genetic modification approach described by Cheah et al., 2012[1] (see Figure 10.1). This method initially mimics a classical gene insertion method as it also is based on the introduction of a positive selection marker (e.g. resistance to kanamycin or spectinomycin). However, this is done along with the insertion of a counter-selection marker (e.g. the toxic gene mazF) into the same chromosomal locus. Then, resorting to a second homologous recombination, both selection markers are replaced by a sequence of interest, resulting in a markerless gene insertion or deletion. All the production strains in this iGEM project were engineered according to this principle and are therefore, considered biosafe and ready to be further engineered or used in a real-world setting.

Batch and Turbidostat cultivation

Introduction

Most of our cultivations are performed in a Multi-Cultivator. “It serves for small scale cultivation of algae, bacteria or cyanobacteria. It consists of 8 test-tubes, each holding up to ca. 85 ml of cultivated suspension. The test-tubes are immersed in a thermostated waterbath (its temperature is controlled) . Each tube is independently illuminated, which is independently adjustable for each test-tube in intensity, timing and modulation. Growth rate of the cultivated organisms may be estimated by automatic measuring of optical density - measured at 680 nm and 720 nm independently at each cultivation tube. Each cultivation tube can be bubbled with air or selected gas (optional) of different flow rate through a manually adjustable valve manifold.”[3]

Biobrick T-vectors

Introduction

BioBrick parts are DNA sequences which conform to a restriction-enzyme assembly standard. Several standards have been developed, using different restriction enzymes and therefore require different materials and methods. In our lab, BioBrick ‘T’ vectors are used for functional block assembling. The system is based on TA-cloning (also known as rapid cloning) and avoids the use of restriction enzymes (fig. 10.3).

References

  • 1. Yi Ern Cheah, Stevan C. Albers, and Christie A M Peebles. A novel counter-selection method for markerless genetic modification in Synechocystis sp. PCC 6803. Biotechnology Progress , 29(1):23–30, 2013.
  • 2. Heidorn, T., Camsund, D., Huang, H. H., Lindberg, P., Oliveira, P., Stensjö, K., & Lindblad, P. (2011). Synthetic biology in cyanobacteria engineering and analyzing novel functions. Methods in enzymology, 497, 539-579.
  • 3. Zhu, T., Xie, X., Li, Z., Tan, X. & Lu, X. Enhancing photosynthetic production of ethylene in genetically engineered Synechocystis sp. PCC 6803. Green Chem. 17, 421–434 (2015).
  • 4. https://www.psi.cz/products/photobioreactors/multi-cultivator-mc-1000