Team:QHFZ-China/Contribution
Contribution
We are lucky to have the chance to get into the lab this year. Hence, to meet the standard of these criteria, we choose the guidance "Add new documentation to an existing Part on that Part's Registry page, new data collected from laboratory experiments." We tested several parts this year, such as promoters BBa_J23100, BBa_J23107, BBa_J23109; RBS BBa_B0034; terminator B0015; green fluorescent protein, GFP, and red fluorescence protein, dsRed. From them, we chose three ones, BBa_J23100, BBa_J23107 and BBa_I0500.
Documentation 1: BBa_J23100 Promoter J23100 and BBa_J23107 Promoter J23107
This year, we quantitatively measured the strength of the promoters J23100, J23107 and J2309 with sfGFP (BBa_K3457015) and CAHS 106094 (BBa_K3457012), and compared with the official data of iGEM. It is convenient for iGEM teams to select better promoters through the data and achieve the best expression level of TDP. We have uploaded the content onto the Registry. Now we will carry the content here for a convenient check.
We measured BBa_J23100, BBa_J23107 and BBa_J23109 as a strong, moderate and weak promoter respectively in 2020. For all the experiments below, we use E. coli BL21(DE3) strain.
Part 1: Measurement with a reporter, sfGFP
Description
First, we measured the strength of the promoter by sfGFP BBa_K3457015.
Protocol
The gene circuit we used is as below:
Figure 1. The Schematic cartoon of the DNA construct to test J23100 / J23107 / J23109 with sfGFP.
The protocol is as below:
(1) Pick clones in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 2mM iPTG into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
(3) The bacteria solution was centrifuged, and the LB medium was removed. Then the bacteria were resuspended by PBS. 100 μL such solution was put into a well of a 96-well plate. The GFP fluorescence and OD600 were detected by microplate readers (Bio-Teck). The parameters are exciting light: 488 nm, light reception: 520 nm, gain 50.
(4) The value of PBS was deducted from the result above. GFP / OD600 was calculated.
Result
Figure 2. sfGFP was expressed with J23100 / J23107 / J23109.
WWe set the strength of J23109 as 1. The relative strengths of J23107 and J23109 were 4.4 and 12.0. Though they are not the same as the data at the top of this part's main page, they worked well, and the strength order of the three promoters was consistent with other people's data. The difference may owe to a certain gene circuit and protocol.
Part 2: Measurement with CHAS 106094
Description
Second, we measured the strength of the promoter by CAHS 106094 BBa_K3457012. This year, we used CAHS 106094 to protect bacteria from freeze-drying and dry storage. We used different promoters to adjust the expression level of CAHS 106094, to study the relationship between the survival rate and CAHS 106094 expression level.
Protocol
The gene circuit we used is as below:
Figure 3. The Schematic cartoon of the DNA construct to test J23100 / J23107 / J23109 with CAHS 106094.
The protocol is as below:
Figure 4. Experiment protocol.
【Day 1】Induction culture
(1) Pick clones in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 2mM iPTG into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
【Day 2】Freeze-dried
(1) If fluorescence induced by the iPTG is detectable in the control group (GFP), continue conducting the
experiment.
(2) Use spectrophotometer to measure the OD600 of the bacteria solution, OD600 = 1
equals to
109 cells. If the OD600 value is between 0.1 and 1, There is a linear relationship
between
OD600 and bacterial density. Calculate the volume of bacterial solution for 109 cells
by using
the formula V = 100 / (OD600 × Dilution ratio).
(3) Take out a measured amount of 109 cells and centrifuge it at 8000 rpm for 3 min. Then pour
out the
supernatant.
(4) Resuspend the bacteria in a 15 mL tube with pre-refrigerated 100 μL 3% glucose solution.
(5) Take off the cover of the tube and put the bacteria into the cold trap. Open the compressor of the
lyophilization machine and freeze the shake tube for 2 h at -70℃.
(6) Put the caky bacteria solution into the drying chamber of the lyophilization machine. Open the vacuum
pump to
dry it in vacuum for 6h at 1 Pa vacuum degree.
(7) Turn off the vacuum pump, place it at seal box filled with silica-gel desiccant a for 2 days at room
temperature.
【Day 3】Room temperature storage
【Day 4】Detect the survival rate
(1) Add 1 mL of sterile water to the tube, vortex for 15 s, placed it at room temperature for 10 min.
(2) Adjust the density of the bacteria solution by gradient dilution, then spread 100 μL of the bacteria
solution on
the LB plate.
(3) If the density above is not suitable, take 100μL of the solution and spread it on the LB plate after
several
gradient dilutions.
(4) Culture the bacteria overnight at 37℃.
【Day 5】Cell Count
(1) Take out the LB plate and take photos to record experimental results.
(2) Use the automatic cell counting function of Image J to count the clone number on the LB plate, then
compare the
results between each group.
Result
Figure 5. The Cfu of bacteria expressing CAHS 106094 after freeze-drying with J23100 / J23107 / J23109.
As expected, J23100 is the strongest promoter and it gave the best survival rate. J23107 is the second and J23109 seemed too weak to express enough CAHS 106094. In conclusion, J23100 and J23107 is effective in this situation, but J23109 is not.
Documentation 2: BBa_I0500 Inducible pBad/araC promoter.
TDPs (Tardigrade intrinsically Disordered Proteins) show protective effect to bacteria. However, related studies are all use T7 promoter and E. coli BL21 (DE3) strain. This year, we used the Inducible pBad/araC promoter to express a TDP, CAHS 106094, in another strain. For all the experiments below, we use E. coli DH5α strain.
Part 1: Measurement with a reporter, GFP
Description
First, we measured the function of the promoter by GFP. We got the gene part from team NEFU-China 2020 as a gift.
Protocol
The gene circuit we used is as below:
Figure 1. The Schematic cartoon of the DNA construct to test BBa_I0500 with GFP.
The protocol is as below:
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to
grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add inducer into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1
to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
(3) 100 μL bacteria solution solution was put into a well of a 96-well palte. The GFP fluorescence and OD600 were detected
by a microplate readers (Bio-Teck). The parameters are: exciting light: 488 nm, light reception: 520 nm, gain: 50.
(4) The value of LB medium was deducted from the result above. GFP / OD600 was calculated.
Note: For step (3), The inducer usually was 0.2% L-arabinose. However, it can be changed if we want to study the concentration of the inducer and the effect of D-arabinose. To study whether glucose and trehalose affect the promoter, we even added D-glucose or D-trehalose of different concentrations with 0.2% L-arabinose into the medium.
Result
Figure 2. GFP can be induced by 0.2% L-arabinose, but not D-arabinose. Glucose and trehalose afected the promoter.
The result is quite clear. First, L-arabinose efficiently induced the expression of GFP. 0.2% arabinose seemed enough. However, D-arabinose did not have any effect. Secondly, glucose and trehalose suppressed the promoter, even though 0.2% L-arabinose was added. At the concentration of 0.3%, the effect of trehalose was even worse than that of glucose. The effect of glucose is well-known. However, that of trehalose is not. We give a hypothesis here. One trehalose can be hydrolyzed into two glucose. So 0.3% trehalose equals to 0.6% glucose. Thus, at a low concentration, trehalose is more unsufferable to the promoter than glucose.
Part 2: Measurement with CHAS 106094
Description
Second, we measured the strength of the promoter by CAHS 106094 BBa_K3457012. This year, we used CAHS 106094 to protect bacteria from freeze-drying and dry storage. We used the promoter to express CAHS 106094 in E. coli DH5α strain, to study whether CAHS 106094 worked in this strain.
Protocol
The gene circuit we used is as below:
Figure 3. The Schematic cartoon of the DNA construct to test J23100 / J23107 / J23109 with CAHS 106094.
The protocol is as below:
Figure 4. Experiment protocol.
【Day 1】Induction culture
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake
them to
grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 0.2% L-arabinose into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in
step 1
to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
【Day 2】Freeze-dried
(1) If fluorescence induced by the iPTG is detectable in the control group (GFP), continue conducting the
experiment.
(2) Use spectrophotometer to measure the OD600 of the bacteria solution, OD600 = 1
equals to
109 cells. If the OD600 value is between 0.1 and 1, There is a linear relationship
between
OD600 and bacterial density. Calculate the volume of bacterial solution for 109 cells
by using
the formula V = 100 / (OD600 × Dilution ratio).
(3) Take out a measured amount of 109 cells and centrifuge it at 8000 rpm for 3 min. Then pour
out the
supernatant.
(4) Resuspend the bacteria in a 15 mL tube with pre-refrigerated 100 μL 3% glucose solution.
(5) Take off the cover of the tube and put the bacteria into the cold trap. Open the compressor of the
lyophilization machine and freeze the shake tube for 2 h at -70℃.
(6) Put the caky bacteria solution into the drying chamber of the lyophilization machine. Open the vacuum
pump to
dry it in vacuum for 6h at 1 Pa vacuum degree.
(7) Turn off the vacuum pump, place it at seal box filled with silica-gel desiccant a for 2 days at room
temperature.
【Day 3】Room temperature storage
【Day 4】Detect the survival rate
(1) Add 1 mL of sterile water to the tube, vortex for 15 s, placed it at room temperature for 10 min.
(2) Adjust the density of the bacteria solution by gradient dilution, then spread 100 μL of the bacteria
solution on
the LB plate.
(3) If the density above is not suitable, take 100μL of the solution and spread it on the LB plate after
several
gradient dilutions.
(4) Culture the bacteria overnight at 37℃.
【Day 5】Cell Count
(1) Take out the LB plate and take photos to record experimental results.
(2) Use the automatic cell counting function of Image J to count the clone number on the LB plate, then
compare the
results between each group.
Result
Figure 5. The Cfu of bacteria expressing CAHS 106094 after freeze-drying.
As expected, the bacteria with an Arabinose promoter (BBa_I0500) and CAHS 106094 exhibited a higher survival rate, which indicated that the promoter and L-arabinose could successfully induce the expression of CAHS 106094 at enough level in E. coli DH5α strain.
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