Big Picture

# Gal4-dependent promoters testing

The graph depicts the fluorescence of three separate liquid cultures of E. coli transformed with plasmids, containing QPAS1-Gal4 repressor and the YFP gene under the designed Gal4-dependent promoter, attributed accordingly. The presence of the plasmid is proven by the fact that the cells grew successfully on the selective medium and the specific YFP fluorescence band was measured.

~|| ![](YFP baseline fluorescence) |
![](YFP fluorescence under QPAS-Gal4 repression) ||

It can be seen that cells containing YFP under Gal4-dependent promoters fluoresce less intensively. Namely, the intensity of YFP under p119_G4 is 3.5 times lower than the control (p<0.05); the intensity of YFP p119_G4_n11 is 7 times lower (p<0.05). 

We suppose that the presence of the different plasmid variants and the drop observed in the fluorescence are correlated. The main hypothesis is that QPAS-Gal4 is expressed and binds to the promoter region.

The remaining fluorescence may be explained by the stoichiometric discrepancies, e.g. the high-copy 4 kb pUC19 outnumbers the 25.2 kDa QPAS-Gal4, and some of the promoters remain unbound. The measurement elaborations might be: to increase the number of QPAS-Gal4 in a cell, either by integration into genome or increasing the relative promoter strength/copy number/RBS strength in QPAS-Gal4. 

> Thus we get two new promoters dependent on Gal4 that can be used in prokaryotic organisms that we consider to be a significant part improvement. Our further step is to try other Anderson promoters to create a library of bacterial Gal4 dependant promoters that there could be more chance to select the best fitting element for different chassis. 

# Recombination locus

To transform our strain of *Arthrospira platenis* we are planning to use homologous recombination in the locus around the proposed gene of resistance to kanamycin. 
To prove check whether genome of our strain contains this gene, we sequenced the proposed DNA sequence. To do so two sets of primers, homologous to parts of kanamycin resistance gene of NIES-39 *Arthrospira platesis*, were ordered. The proposed gene of NIES-39 can be found in GeneBank. This was followed with amplification and sequencing of proposed kanamycin resistance gene in IPPAS B-256. 

![](Electrophoresis of amplified sequence with 2 combinations of primers)
Sequence analysis has shown that the sequence we are dealing codes two particles of one protein, which belongs to the family of phosphotransferases. This protein has not been annotated yet and its biochemical features are yet little uncovered.
> The identity of the gene sequence in *Arthrospira platensis* IPPAS B-256 and the sequence of *Arthrospira platensis* NIES-39 in GeneBank accounts at 99% (e-value = 0.0, Per. Ident = 90.23%). Therefor, we can suppose that the identity of the flanking sequences can be also that high. This increases our chances of a successful transformation.

We also analysed homologous sequences in other cyanobacteria. 
> It was determined that identical sequences can be observed not only in *Arthrospira*, but also in other families *Oscillatoriales* and some families *Nostocales*.

# Characterization of Arthrospira platensis 
(Nowicka-Krawczyk, Mühlsteinová & Hauer IPPAS B-256)


##  Overview
1.	Name of the organisation: Institute of Plant Physiology of Russian Academy of Science (IPPRAS)

2.	Author name of the strain, code: К Leonard, 1968. 

3.	Date of strain isolation: 1968 г.

4.	Other names: *Spirulina platensis* (Gomont) Geitler; *Arthrospira platensis* (Nordstedt) Gomont

### Nomenclature 
**Phylum:** *Cyanobacteria*

**Class:** *Cyanophyceae*

**Order:** *Oscillatoriales*

**Family:** *Microcoleaceae*

**Genus:** *Limnospira* 

**Species:** *Arthrospira platensis*

**Identified:** Sinetova M.A., 2019.

**Isolated from:** Republic Chad, natron lake Bodou. 

**Nucleotide sequences in GenBank:** KX262886

(M.D. Guiry in Guiry, M.D. & Guiry, G.M. 2020. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway.; searched on 18 December 2020).

### Axenicity
**Purification rate:** contaminated with bacteria

**Control method:** test in Zarrouk medium with addition of 0,2 % glucose and 0,02% casein hydrolysate; microscopy. 

##  Physiological characteristics 

### Productivity in optimal cultivation condition
**We have measured some cultivation culture parameters in intensive cultivation conditions:**
1. average rate of biomass accumulation in linear phase at high density of cultures, v mean. (measured corresponding to dry weight, g/l per 24-hour period): **1,21**
2. relative growth rate in linear phase at high density of cultures, μ (per 24-hour period), evaluated according to the optical density changes at 750 nm: **0,301** ; evaluated according to dry weight changes: **0,317**
3. maximum relative growth rate in exponential phase, μmax (per 24-hour period): **1,195** (time of biomass duplication — **13,9 h**).
4. final biomass concentration (g of dry weight / l): **9,5±0,1** 

> As we see, IPPAS B-256 shows high growth rates in intensive cultivation conditions.

### Biochemical content
**We have conducted a biochemical analysis of IPPAS B-256 culture:** 

| Cultivation condition | Chlorophyll *а*, mg/g of dry weight | Carotenoids, mg/g of dry weight | Chlorophyll *a* ÷ carotenoids |
| Exponential phase | 13,6±0,4 | 4,2±0,2 | 3,25 |
| Stationary phase  | 3,7 ±0,4 | 1,1 ±0,1 | 3,4 |
*Table 3 – Pigment content*

| Cultivation condition | Protein, mg/g of dry weight | Lipids, mg/g of dry weight | Storage carbohydrates*, mg/g of dry weight |
| Exponential phase | 254±28 | 45±3 | 65±8 |
| Stationary phase | 163±14 | 30±1 | 135±10 |
*Table 4 – Protein, lipid and storage carbohydrates content*
* — Mainly glycogen 

| Fatty acids | FA content (mol %) | |
|  | Exponential phase | Stationary phase |
| 16:0 | 43,9±1,3 | 50,0±1,0 |
| 9-16:1 | 2,7±0,0 | 3,5±0,3 |
| 18:0 | 19,0±2,8 | 8,3±1,4 |
| 9-18:1 | 9,3±0,3 | 12,0±0,4 |
| 9,12-18:2 | 14,6±0,4 | 17,3±0,4 |
| 6,9,12-18:3 | 10,6±0,9 | 8,9±0,4 |
| Total lipids | 0,730 | 0,767 |
*Table 5 – Fatty acid content*

### Sustainability of the culture 
1. **seasonal fluctuations:** not observed
2. **resistance to contamination:** displaces contaminants in intensive cultivation condition
3. **autolysis:** not observed
4. **aglutination:** not observed, however, conglomerates of strands are constantly formed, appropriate mixing regime is required.

## Biotechnological characterisation

All in all, here are some biotechnological tendencies we collected which IPPAS B-256 cultivation:

-  High productivity
-  High protein content
-  High growth rate during a significantly long period of time (4 days) in intensive cultivation conditions
-  No lag-phase in the beginning of cultivation is observed
-  A proper mixing regime is required in order to prevent formation of strands aggregates.

> We believe, that this properties of the strain make it a suitable candidate for further biotechnological research and, particularly, our purposes. 

We have also made a biochemical analysis of our strain and revealed that its biomass consist of
- Lipids (8.2%)
- Carbohydrates (15.8%)
- Proteins (62.2%)
![](Composition of 105 mg of IPPAS B-256 biomass (mg))

> This way we prove that our strain of *Arthrospira platensis* is as rich in proteins (> 60%) as it it stated in literature.

## Culture while nitrogen and phosphorus starvation

> Nitrogen and phosphorus starvations were chosen as abiotic stress conditions which often initiate accumulation of storage products, such as starch/glycogen or lipids, and secondary metabolites. Therefore, **we chose N and P starvations as stress conditions** in order to investigate **carbon storage strategies and metabolic adaptation** of *Arthrospira platensis* 
> As it can be seen in the diagram, IPPAS B-256 is **not tolerant to N starvation**, while **P starvation decreases growth rate, but does not lead to culture degradation**. This experiment had triple replication and lasted 7 days. Optical density at λ=750 nm and λ=680 nm was measured on the 1st and on the last day of the experiment.
OD750 represents overall cloudiness of probe and this data corresponds to an approximate order of cells. OD680 represents data of chlorophyll *a* reflection and can also be interpreted as approximate order of cells in the probe. 

> We also investigated carbon storage in the form of triacylglycerols in IPPAS B-256 cells. On the 7th day of experiment probes for light and fluorescent microscopy were collected, so that culture condition and form of lipid (TAG) storage could be observed. Probes were dyed with lipophilic fluorescent dye BODIPY (green). Moreover, autofluorescence of chlorophyll was stimulated (red).

| Control |
| ![] |
| ![] |
| Cells are in the active proliferation phase. There are numerous TAG drops in the cytoplasm along thylakoids. |

| –N |
| ![] |
| ![] |
| Many cells were exposed to degradation. TAG drops are almost not observed. |

| -P |
| ![] |
| ![] |
| Cells store less TAGs than in the control cultures. TAG drops are observed mainly along septum between to cells in the strand. |
> In laboratory conditions *Arthrospira platensis* cells often quit helix formation. We have revealed, that **during phosphorus starvation condition cells began helix formation**, however, with a bigger diameter than in cells in nature.