Team:OUC-China/proof3

proof3

Mini system

We set four different permutations for our mini promoter and mini terminator thus constructed four dif-ferent circuits. For each one, the strength level of promoters are characterized by yECitrine, a kind of yel-low fluorescent protein. And we use red fluorescent protein mStrawberry to represent the read through of terminators.

In the wet lab study, we measured both the expression level of YFP and RFP to validate the expected performance of the MINI system in comparation of normal promoters and terminators whose expression level are measured as well.

Meanwhile, we monitored the growth rate of the recombinant yeast to specify the expression of MINI system at a particular living stage. Moreover, we conducted qPCR towards corresponding protein in or-der for a further validation of MINI system’s strong expression on a post-transcriptional level.

We also expend the application range of this system by contact with other teams and test it in different yeast strains and experimental environment, which can also be an important part of our collaboration. the MINI system has a high performance ratio in driving gene expression. The obvious advantage of the MINI system is to provide a simple model for studying promoter mutations and promoter modifications.

Circuit construction

We synthesized minip, minit, cyc1p, cyc1t and built four circuit with Gibson assembly by arranging them in different orders. The plasmids were then imported into yeast EBY100.

Fig 3.1 The plasmid map of our circuit.

Fig 3.2

Function verify

We characterize the strength of the MINI system by detecting the fluorescence intensity of the yeCitrine. We measured the growth curves of four strains containing different expression systems, and measured the intensity of excitation and emission of yeCitrine, respectively 502nm and 532nm.

For convenience, we named the “Pmini-yECitrine-Tcyc1-mStrawberry-Tcyc1” as “mc”, “Pmini-yECitrine-Tmini-mStrawberry-Tcyc1” as “mm”, “Pcyc1-yECitrine-Tcyc1-mStrawberry-Tcyc1” as “cc” and “Pcyc1-yECitrine-Tmini-mStrawberry-Tcyc1” as “cm” hereafter.

Fig 3.3 The strength of the MINI system.

The fluorescence intensity is: mm> cm> mc> cc, in which mm circuit has the highest expression, proving our successful construction of MINI system. To draw a conclusion, the system has a strong expression, but a very short nucleotide sequence. In this ex-ample, the combination of weak promoter + strong terminator is better than that of strong promoter + weak terminator.

With the help of the other two teams, we completed the repeated testing of the MINI system. The time of the test data is the late logarithmic phase of yeast growth. The yeast strains we used with NJU-China were Saccharomyces cerevisiae EBY 100. The yeast strain used in TJU China was Synthetic yeast synX.

Fig 3.4 MINI system fluorescence measurements from three teams, OUC-Chian,TJU-China, NJU-China (left to right)

Through the fluorescence intensity map, we have not yet clear the true advantage of MINI system. But from the following figure, we can directly observe the relationship between a nucleotide base number and fluorescence intensity. The red arrows indicate the short and strong features of the mm gene circuit.

Fig 3.5 Promoter and terminator of the nucleotide base length and intensity. (For example, mm represents the base length of Pmini + Tmini).

In order to verify that the MINI system was able to work in a variety of yeast strains, we invit-ed other teams to conduct a repeat experiment. Especially in TJU-China, the use of their own synthesis of yeast to complete this experiment also verified the MINI system with versa-tility.

Validation of expression on transcription level

The result of qPCR shows that at 22^nd hour the system reached the highest expression intensity and the expression of four circuits is shown below. The error bars indicate s.d. of mean of exper-iments in triplicate.

Fig 3.6

In the chart, the RNA content of yECitrine comes as the following order: mm>mc>cm>cc, which is not completely consist with the result of protein level. mm’s RNA content is several times that of cc. Compared to Figure b, this difference in RNA content does not reflect the protein content very well, yet still, our MINI system has an obvious superiority over normal combination (cc), which confirmed our hypothesis. As shown above, generally the magni-tude of leakage is over 100 times smaller, so it can be ignored to some extent.

To draw a conclusion, mm has the smallest size and the strongest expression with a leakage that can almost be ignored. What’s more our system functions well in different strains and experimental conditions, which proves its potential to apply in various situations.

PROTOCOL

Reserve transcription

Material
PrimeScript^™RT reagent Kit with gDNA Eraser (TaKaRa Code No. RR047A)
Procedure
1. Genomic DNA elimination reaction
1) Prepare the genomic DNA elimination reaction solution on ice.
2) Add RNA template with the suitable amount of the master mix to a PCR tube.

Reagent	Amount
5X gDNA Eraser Buffer	2.0 μl
gDNA Eraser	1.0 μl
Total RNA	1.0 μl(Up to 1 μg of total RNA)
RNase Free dH₂O	Up to 10.0 μl

3) Run the program: 42℃ 2 min
2. 4℃ Reverse-transcription reaction
1) Prepare the reverse-transcription reaction solution on ice.
2) Add Reaction solution from Step 1 with the suitable amount of the master mix to a PCR tube

Reagent	Amount
Reaction solution from Step 1	10.0 μl
5X PrimeScript Buffer 2 (for Real Time)	4.0 μl
RT Primer Mix	4.0 μl
PrimeScript RT Enzyme Mix I	1.0 μl
RNase Free dH₂O	4.0 μl

3) Run the program:
37℃15 min
85℃ 5 sec
4℃

Total RNA Extraction

Material
RNAiso Plus(Takara Co.9109)
Procedure
1. Cleavage of yeast cells.
1） Take 1 ml OD600 value of 1.5 to 2.5 liquid culture yeast to 1.5 ml Microtube and 8,000 g at 4 ° C for 2 min.
2） carefully discard the supernatant, slowly add 1 ml of ice to the precipitation of sterile water, with a pipette gently blowing, so that precipitation resuspended.
3） 8,000 g at 4 ° C for 2 min. Carefully discard the supernatant, as far as possible in addition to net liquid.
4） Add 0.4 ml of Yeast RNAprep Buffer to the precipitate and gently re-blow with a pipette to resuspend the pellet.
5） into the 30 ° C water bath for 1 hour, during which gently shake the centrifuge tube 1 or 2 times.
6） Remove the centrifuge tube from the 30 ° C water bath and centrifuge at 12,000 g for 4 minutes at 4 ° C.
7） Carefully discard the supernatant, add 1 ml of RNAiso Plus to the precipitate, gently blow with a pipette to resuspend the pellet.
8） cover the centrifuge tube cover, whirlpool oscillation 2 to 5 minutes to clarify the suspension. 12,000 g at 4 ° C for 5 min.
9） Carefully aspirate the supernatant and move into a new 1.5 ml RNase-free Microtube (do not touch the precipitate).
2.Collection
1) Collect and pipettes 1.5~2.5ml bacteria which is in the log phage(usually when OD600=1.0) into a centrifuge tube. Centrifuge tube for 8,000×g,5 minutes at 4°C.Discard supernatant and be care not to disturb the bacteria pellet.
2) Add 1ml of RNAiso Plus, pipette up and down until pellet is completely resuspended.
3) Leave at room temperature(15~30°C) for 5 minutes, isolate the RNA from the nuclear protein.
2.Extracion of total RNA
1) Add 200 ul chloroform, cap the centrifuge tube and mix until the solution becomes milky.
2) Keep the solution at room temperature for 5 minutes.
3) Centrifuge at 12,000×g for 15 minutes at 4°C.Centrifuging the solution will separate it into three layers; liquid top layer(contains RNA),semisolid middle layer(mostly DNA),and bottom organic solvent layer.
4) Transfer the top liquid layer to new centrifuge tube without touching middle layer.
5) Measure the amount of the top layer and add an equal amount or add up to 0.5 times of isopropanol of the top layer. Mix together well. Keep the mixture at room temperature for 10 minutes.
6) Centrifuge at 12,000×g for 10 minutes at 4°C to precipitate the RNA.
7) Cleaning RNA precipitate
8) Carefully remove the supernatant, do not touch the pellet.  9) Add an amount of 75% cold ethanol that was equivalent to the supernatant. Clean the precipitate by vortexing.
10) Centrifuge the solution at 7,500×g for 5 minutes at 4°C and discard supernatant. Be care not to disturb the precipitate.
4.Dissolving RNA
Dry the precipitate by leaving the tube open for several minutes. After the precipitate is dry, dissolved it with appropriate amount of RNase-free water.
Attention
Make sure that all the centrifuge tubes and pipettes have been treated with DEPC.

Quantitative Real-time PCR

Material
SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa(Code No. RR420A)
Procedure
1. Prepare the PCR mixture shown below

Reagent	Volume	Final conc.
SYBR Premix Ex Taq (Tli RNaseH Plus) (2X)	10 μl	1X
PCR Forward Primer (10 μM)	0.4 μl	0.2 μM
PCR Reverse Primer (10 μM)	0.4 μl	0.2 μM
Template (< 100 ng)	2 μl
dH2O (sterile distilled water)	7.2 μl
Total	20 μl

2. Start the reaction using LightCycler 480 System
1) Denature:
95℃ 30 sec. (Ramp rate: 4.4℃/sec.)
1 cycle
2) PCR :
95℃ 5 sec. (Ramp rate: 4.4℃/sec.)
60℃ 30 sec. (Ramp rate: 2.2℃/sec.)
40 cycles
3) Melting
95℃ 5 sec. (Ramp rate: 4.4℃/sec.)
60℃ 1 min. (Ramp rate: 2.2℃/sec.)
95℃ (Ramp rate: 0.11℃/sec.)
1 cycle
4) Cooling
50℃ 30 sec. (Ramp rate: 2.2℃/sec.)
1 cycle
3. After the reaction is complete, check the amplification and melting curves and plot a standard curve if absolute quantification will be performed.