Part:BBa_K2136010

5' cassete for Chlamydomonas transgenic expression

Synthethic cassette for algae expression.

This composite part is the 5' part of a system to express proteins in Chlamydomonas reinhardtii. It includes a promoter, a resistance gene with RbcS introns 1 and 2, a self cleaving peptide and a signal peptide. This combination, in conjunction with a in-frame coding sequence and a microalgae terminator enables the efficient translation and secretion to the growing media of the constructs of interest.

Explanation of the individual parts:

BBa_K2136013

AR1-Rbcs2

Constitutive promoter for use in Chlamydomonas reinhardtii based on the fusion of DNA sequences upstream of Heat Shock Protein 70A (nucleotides 1-275) and rbcS2 (nucleotides 294-474) genes. This part also includes a 5'-UTR derived from the rbcS2 gene.

BBa_K2136014

Resistance gene for algae expression. Bleomycin gives a resistance against zeocin, because ble binds to zeocin blocking its function, Bleomycin have to introns inserted in its coding sequence.

Figure 1: Gel electrophoresis of mCherry+pSB1C3 construct

After ligating the sequence in the pJP22, we wanted to find the best way to transform C. reinhardtii, we tested a proprietary buffer, TAP medium and water as buffers during the electroporation. Then, cells were plated in Agar-TAP medium. Petri dishes with different concentrations of Zeocin (an antibiotic from Bleomycin family) because the plasmid used had a sequence for Bleomycin resistance.

           Figure 2: pJP22 mCherry transformants - Zeocin 5 ug/mL                    Figure 3: pJP22 mCherry transformants - Zeocin 10 ug/mL

        Figure 4: pJP22 mCherry transformants - Z5 (Water Transformed)                Figure 5: pJP22 mCherry transformants - Z10 (Water Transformed)
       Figure 6: pJP22 mCherry transformants - Z5 (TAP Transformed)               Figure 7: pJP22 mCherry transformants - Z10 (TAP Transformed)
pJP22 Figure 8: mCherry transformants - Z5 (Sapphire Transformed)                  Figure 9: pJP22 mCherry transformants - Z10 (Sapphire Transformed)

As expected, the number of colony-forming units is much higher in the media with less Zeomicin.

At the same time, the transformation of cells were not so good using the proprietary buffer in comparison with Water and TAP medium.

This part was characterized through the expression of a codon optimized version of mCherry reporter protein (https://parts.igem.org/Part:BBa_K2136016). We successfully amplified the codon optimized mCherry, which has 711 bp, as can be seen in Figure 1.

Figure 1: Gel electrophoresis of mCherry+pSB1C3 construct

After ligating the sequence in the pJP22, we wanted to find the best way to transform C. reinhardtii, we tested a proprietary buffer, TAP medium and water as buffers during the electroporation. Then, cells were plated in Agar-TAP medium Petri dishes with different Zeocin concentrations (an antibiotic from Bleomycin family) because the plasmid used had a sequence for Bleomycin resistance.

After 7 days we selected clones from previous dishes and started new cultures in a 96 well plate with 200 uL of TAP medium per well, agitation of 800 rpm, 25°C +- 1°C and 80 μE m−2 s−1 luminosity and a clear film sealing the plate. We also filled some wells with wild C. reinhardtii, just TAP medium or just mCherry as controls.

Figure 2: Cultivation setup for screening

For a better characterization of mCherry we’ve measured its excitation and emission spectra using a Tecan M200 Pro Microplate reader. In the 96 well plate we’ve measured the excitation and emission spectra of transformed C. reinhardtii supernatant, wild C. reinhardtii supernatant, water, TAP medium, transformed C. reinhardtii with spent TAP, wild C. reinhardtii with spent TAP, washed transformed C. reinhardtii with fresh TAP and washed wild C. reinhardtii with fresh TAP. For mCherry fluorescence detection we used excitation wavelength at 575 nm and emission at 608 nm, for inactive mCherry we used excitation wavelength at 410 nm and emission at 461, for Chlorophyll fluorescence we used 440 nm for excitation and 680 nm for emission. We also measured the absorbance at 750 nm for cellular concentration.

The TOP 5 mCherry-producer clones were E 10, which was transformed with TAP medium, and B1, B5, B6 and B11, which were transformed with a proprietary buffer as can be seen in Figure 3.

     Figure 3: TOP 5 mCherry producers in the first screening          Figure 4: TOP 5 mCherry producers in the second screening

Before purifying we wanted to see by our own eyes that mCherry was present in our samples. In order to do that, we used a fluorescence microscope. mCherry is shining bright like a diamond in the sky in Figure 5 and 6.

Figure 5: Fluorescence microscopy of C. reinhardtii. A - Measuring mCherry fluorescence. B- Measuring chlorophyll fluorescence. C - Open field image. D - Superposition of A,B and C.


Figure 6: 3D Fluorescence microscopy of C. reinhardtii. Chlorophyll is fluorescing in green and mCherry is fluorescing in red.

Besides the previous method, we also wanted to see mCherry present in the cell’s supernatant. For that, we made the following qualitative analysis schematized in Figure 7:

Figure 7: Experimental setup for qualitative detection of mCherry.

This special filter is able to block the laser light and at the same time allows the light emitted by mCherry to pass through it, as shown in Figure 8.

Figure 8: Spectra of experimental setup components

Our results are shown below in Figures 9.

Figure 9: Laser passing through cellular supernatant. A - Laser is passing through a wild type C. reinhardtii supernatant. B- Laser is passing through a transformed C. reinhardtii producing mCherry.

So we achieved to implement an efficient protein expression and secretion system for Chlamydomonas for the first time in iGEM!! Corroborating, one more time, to our proof of concept.

We used Fast Protein Liquid Chromatography (FPLC) to analyse and purify our mCherry. FPLC was used with an Ion exchange column that exploit the net electrostatic charges of proteins, in pH values different of their pI (Isoelectric point). We developed a purification protocol to mCherry. First, we performed a gradient purification to establish the best salt concentration to elute mCherry.

Gradient Set Up:

Column: Resource Q (6 mL) - GE Healthcare
Buffer A: Sodium Phosphate 50 mM, pH7.5
Buffer B: Sodium Phosphate 50 mM, pH7.5 + 1M NaCl
Equilibration: 2 column volume (CV)
Injection: 0.5mL 40X Concentrate supernatant sample
Gradient length: 20 CV
Flow rate: 5mL/min
Fractionation: 5mL to unbound and 3 mL to the rest of the method

We obtained the following result (Figure 10).

Figure 10: Chromatogram of gradient mCherry purification. Green line (-) is the UV sensor reading. Red line (-) is buffer B percentage in the mixture. Black line (-) is the conductivity measurement. Blue line (-) is the fluorescence measurement of fractionated samples.

UV absorbance curve integration allow us to estimate the amount of protein separated from mCherry, 99% of all protein detected by the sensor was separated from mCherry fractions.

To further develop our method and reduce processing time, we developed a step based purification method (Figure 2). We kept 0% of B after injection for 3 CV, increase it a little bit to 0.7% of B to try to remove mCherry in this fraction, followed by a 100% of B step. This strategy was performed in a slower flow rate (3mL/min), and allow us to separate mCherry from 2 peaks in the beginning of the method. mCherry still left in the 0% step, but this method proved to be efficient, 99,7% of detected proteins were separated from mCherry.

Step based purification Set Up:

Column: Resource Q (6 mL) - GE Healthcare
Buffer A: Sodium Phosphate 50 mM, pH7.5
Buffer B: Sodium Phosphate 50 mM, pH7.5 + 1M NaCl
Equilibration: 2 column volume (CV)
Injection: 0.5mL 40X Concentrate supernatant sample
Step1: 3 CV
Step2: 2 CV
Step3: 5 CV
Flow rate: 3mL/min
Fractionation: 5mL to unbound and 1 mL to Step1, 3 mL to Step 2 and 5 mL to step 3.
We obtained the following result (Figure 11).
Figure 11: Chromatogram of step based mCherry purification. Green line (-) is the UV sensor reading. Red line (-) is buffer B percentage in the mixture. Black line (-) is the conductivity measurement. Blue line (-) is the fluorescence measurement of fractionated samples.

The samples purified from this method were used to further characterize our mCherry produced by Chlamydomonas reinhardtii. The Excitation/Emission spectrum (Figure 12) obtained are similar to the ones available to mCherry.

Figure 12: Excitation/Emission spectrum of mCherry produced and purified from Chlamydomonas supernatant.

BBa_K2136017 The foot-and-mouth disease virus (FMDV) self cleaving peptide (VKQTLNFDLLKLAGDVESNPGP) is capable of undergoing self-cleavage in vitro, thus separating 2 different in-frame peptide sequences. (1) Another peptide of the family, P2A, was more in-depth described and characterized by iGEM14_Warwick (https://parts.igem.org/Part:BBa_K1442039#)

BBa_K2136018

This sequence is part of the arylsuflatase 2 (Ars2) gene in Chlamydomonas reinhardtii. It is responsible for allowing the secretion of the fused protein to the culture medium, thus allowing cell-lysys independent purification of proteins and/or secretion for other aplications This part also includes a 3' scar with XhoI and ClaI restriction sites included in original gene synthesis to better suit team USP_UNIFESP 2016's workflow.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 264
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1539
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1283
  Illegal NgoMIV site found at 1344
  
• 1000
  COMPATIBLE WITH RFC[1000]