Team:Ionis Paris/Contribution
Contributions
Use of Shewanella oneidensis and measurement of its activity
As a contribution, here is a full description of the utilisation of the bacteria Shewanella oneidensis (S. oneidensis) and how measure its electrical activity.
We mostly inspire our work from a recent paper working with a bioelectronic system with S. oneidensis [1].
S. oneidensis is anaerobic facultative, we can therefore cultivate it with or without the presence of oxygen, at 30°C in LB (Lysogeny broth) media. To make sure that no contamination occurs, we plate 50 mL of liquid culture on LB Agar. As a result, we expect salmon-pink colonies.
Figure 1: Colonies of S. oneidensis
Before using S. oneidensis for testing its electrical activity, we centrifuge our tube at 3000g for 10 minutes to find a salmon-pink pellet to be sure of the quality of our culture.
Measurement of the electrical activity !
To measure the activity of S. oneidensis, we established a protocol to have consistent results through our experiments.
Our system consists in two drilled 50mL Falcon tubes stuck together by a clamp and separated by a NafionTM membrane. We drilled the tubes with a white-hot scalpel, heated with Bunsen burner. The whole process is done around the Busen burner to keep it in a sterilised environment. We then placed our system under the Biosafety cabinet (BSC).
Figure 2: Bioelectronic system
On the right, S. oneidensis in LB. On the left just LB.
In one of the Falcon, we have S. oneidensis cultivated in LB and in the other one there is just LB. In this way we can measure the potential difference between the cathode and the anode (corresponding to 3*3cm2 carbon cloth).
In order to have the same quantity of S. oneidensis for each experiment, we measure the optical density (OD) of our culture. We standardized our measure with a bacterial OD of 0,6 set at 600nm (wavelength used for bacterial density). In each falcon there is a volume of 45 mL of media. For the one with the bacterial culture, we put first 40 mL of LB media and then add 5 mL of bacterial culture at absorbance 0,6 OD600.
To test the activity depending on the level of lactate, we add different quantities of lactate and let incubate for 500 minutes before analysing results. The quantity tested in the article developed by Zeng et al. [1] was 20mM in basal media. We therefore decided to start our experiments with this quantity of lactate in our media.
Preparation of a pSB1A3 backbone for Golden Gate Assembly using Esp3I
During our project, Golden Gate Assembly was our initial method for cloning our plasmids. Even though we did not manage to do so, we did assemble a Biobrick RFC[10] compatible backbone, usable for Golden Gate Assembly using BsmBI/Esp3I (enzymes are isoschizomers).
The idea behind this part was to modify a pSB1A3 containing a RFP (BBa_J04450), our main backbone for restriction enzyme clonings used in our lab, in order to obtain a vector usable both for Restriction enzyme cloning and for Golden Gate Assembly using BsmBI/Esp3I. To do so, we ordered a 121 synthesized fragment from IDT containing random nucleotides and 4 specific sites :
Figure 1 : Ordered fragment with the different wanted cut sites (SnapGene representation)
As shown on Figure 1, the fragment contains Prefix (XbaI) and Suffix (SpeI) restriction sites for insertion inside a Biobrick RFC[10] compatible backbone.
Figure 2 : Esp3I recognition and cut sites (New England Biolabs)
Figure 2 shows the recognition and cut sites of Esp3I. In order to perform Golden Gate Assembly, Esp3I sites have to be oriented in a specific way :
- Site on the left needs to have the following sequence for the top strand : 3’ (N)₅GAGACG 5’
- Site on the right needs to have the following sequence for the top strand : 3’ CGTCTC(N)₅ 5’
When obtained, DNA was resuspended and both the pSB1A3_BBa_J04450 and the DNA fragment were digested with XbaI and SpeI and ligated using a T4 DNA ligase. E. coli DH5-α were transformed using the obtained DNA and the construction was verified using PCR on colonies. For more information on this part, see Results.
Figure 3 : Different uses for the newly form pSB1A3 backbone
As shown on Figure 3, our new backbone can be used for Restriction Enzyme cloning or Golden Gate Assembly. In the context of our project, this backbone was also used as a template for Circular Polymerase Extension Cloning.
Sources
[1] Zeng, J., Banerjee, A., Kim, J. et al. A Novel Bioelectronic Reporter System in Living Cells Tested with a Synthetic Biological Comparator. Sci Rep 9, 7275 (2019). https://doi.org/10.1038/s41598-019-43771-w