Team:Lambert GA/Improve

TUNING SAMPLING

Workflow Summary

Lambert iGEM’s Tuning experiment is modified and adapted from Carolina Science’s Biobuilder iTune Device Lab. Our workflow follows the basic steps of streaking plates with the respective bacteria strains and inoculating in LB liquid cultures, but then it deviates from Biobuilder’s protocol. Instead of taking cells from the liquid cultures and beginning the reactions with ONPG, we subcultured each LB liquid culture into an M9 liquid culture. M9 media is clear, allowing for visualization of the expression of yellow color. After letting the bacteria grow in M9, we took some cells and added ONPG. Rather than lysing the cells, which we found leads to variability in results, we let ONPG diffuse across the cell membrane and be broken down by β-galactosidase (β-gal) inside the cell. Because β-gal remained inside the cell, bicarbonate buffer and carbonate were not needed to regulate its activity. We waited 24 hours, measured absorbance of samples at 420 nm and 600 nm using a plate reader, and inputted the values into the Miller unit formula to calculate β-gal activity, which is proportional to expression.

Wet Lab Protocol

Materials

    • 640 μL of 0.1 M IPTG (Isopropyl-β-D-thiogalactoside)
    • 640 μL of ampicillin
    • 320 ml of sterile LB media
    • 320 ml of sterile M9 media
    • 8 ml of 1X ONPG (ortho-Nitrophenyl-β-galactoside)
    • 40 mg into 40 mL of dH20 → 0.01% solution

Streak Plates

  1. Culture tubes of bacteria strains are ordered from Biobuilder Carolina Laboratories.
  2. Sterilize inoculating loop and top of opened bacteria tube by passing through Bunsen burner flame.
  3. Allow the inoculating loop to cool for a few seconds before inserting into a bacterial culture tube and scraping off a bit of bacteria.
  4. Streak onto the corresponding plate.
  5. Repeat steps a through d for each plate/bacterial strain.
  6. Incubate for 24 hrs at 37°C.

Liquid Cultures

  1. Add 320 μL of amp and 320 μL of IPTG to 320 mL of LB, then aliquot 4 mL of this mixture to each liquid culture tube (80 tubes total).
  2. Add 1 colony from the desired bacterial strain plate to 20 μl of MilliQ water, making sure to pipette up and down to the first stop until the colony is dissolved.
  3. Pipette 5 μL of bacterial mixture into each corresponding liquid culture tube.
  4. Incubate at 37°C for 24 hours, shaking.

Subcultures

  1. Add 320 μL of amp and 320 μL of IPTG to 320 mL of M9 and mix.
  2. Add 4 mL of mixture to each liquid culture tube.
  3. Take 200 μL of cells from the LB liquid cultures and add to the corresponding M9 liquid culture tube.
  4. Incubate at 37°C for 4 hours, shaking.

Reactions

  1. Add 200 μL of cells from the subcultures into small culture tubes.
  2. Create blanks using 200ul of M9 instead of cells.
  3. Add 100μL ONPG into each culture tube.
  4. Add 100μL of ONPG into the blanks as well.
  5. Let all tubes, including blanks, sit for 24 hours in the incubator at 37°C.

Testing

  1. Pipette 200μL of the cell and ONPG mixture for each bacterial strain into its own well in a 96 well plate.
  2. Pipette the blank into one well.
  3. Measure absorbance for each well at 420 nm and 600 nm.

Results

Strain Number

Promoter Part

RBS Part

Promoter/ RBS Description

Miller Units (8 replicates)

1

BBa_J23113

BBa_B0031

Weak/ Weak

5.201

3.358

7.308

6.319

7.467

8.548

5.97

6.054

2

BBa_J23113

BBa_B0032

Weak/ Medium

4.945

5.182

5.149

5.37

5.953

7.462

5.002

2.759

3

BBa_J23113

BBa_B0034

Weak/ Strong

5.268

5.939

5.96

4.329

10.852

10.259

7.233

3.264

4

BBa_J23106

BBa_B0031

Medium/ Weak

5.367

6.092

6.728

5.9

6.437

7.607

7.19

3.247

5

BBa_J23106

BBa_B0032

Medium/ Medium

5.062

5.492

5.798

5.551

6.114

6.646

5.953

8.249

6

BBa_J23106

BBa_B0034

Medium/ Strong

5.332

6.473

6.064

5.695

5.959

7.727

6.409

6.974

7

BBa_J23119

BBa_B0031

Strong/ Weak

6.295

6.46

6.032

6.108

10.367

8.507

6.217

9.444

8

BBa_J23119

BBa_B0032

Strong/ Medium

5.538

6.343

6.536

6.101

9.34

8.407

7.525

8.454

9

BBa_J23119

BBa_B0034

Strong/ Strong

5.97

7.835

13.293

7.711

7.735

13.38

8.64

8.353

Reference

BBa_J23115

BBa_B0035

Reference/ Reference

5.636

 7.034

 6.191

 6.612

 7.485

 7.290

 6.974

 9.421


Combinations 1-9 and R show a prominent yellow color from the ONPG broken down by β-galactosidase to produce ONP. As the promoter strength increases, the yellow color becomes more visible. Combinations 1 and 2 have a clearer color, indicating a weak promoter.

Conclusion

Lambert iGEM’s Tuning experiment supports the change in promoter strength for this year’s toehold construct. Last year, Lambert iGEM utilized the BBa_J23100, a strong promoter that causes overexpression. While constructing this year’s Toehold Biosensor for C. elegans, we decided to use a different promoter to prevent this leakiness. Testing supported our claim that the use of a medium promoter and strong RBS produced the most consistent expression. As a result, this year’s toehold design used a moderate promoter, BBa_J23106, rather than a strong promoter, BBa_J23100.

Using the data from Lambert iGEM’s Tuning experiment, a model was created to predict protein expression based on promoter and RBS strength by inputting promoter and RBS sequences. To represent how the strength of the promoter impacts expression, we built a multivariate linear regression of the data.

As seen in Trial Conclusions below, Lambert iGEM’s Tuning experiment encountered many issues with variability, similar to other iGEM teams conducting enzymatic experiments. To combat this, our lab decided to remove the lysing step from our original protocol from Biobuilder, as the number of lysed cells and the exposed enzymes can vary greatly as a result from lysing. Additionally, our lab decided to allow the experiment to run longer to allow ONPG to cross the cell membrane and react within the cell. These findings, along with our adaptations, can aid other labs and iGEM Teams to combat variability in enzymatic expression experiments.

IMPROVED PART

Overview

In order to illustrate the toehold switch mechanism of the C. elegans biosensor switch, Lambert iGEM synthesized a standard toehold switch construct that was originally designed by the Collins Lab to serve as the proof of concept for the comparison of the reporter gene expression of the Green Fluorescent Protein (eGFP). Part BBa_2550000 was the model for this year’s design however, it had a flaw of overexpression. To engineer a better toehold, Lambert iGEM designed two composite parts to test our promoter choices; BBa_J23100 and BBa_ J23106. These new GFP toehold switches allowed us to quantify the control of the system by using the industry standards of plate readers and our own Fluorometer.

Assembly

In order to induce a strong expression of the downstream reporter, the 2018 team used a strong T7 promoter (BBa_J23100) from the Anderson Promoter family. Promoter BBa_J23100 had a reported strength of 1, making it the strongest promoter of the family barring the consensus strain. However, the construct experienced overexpression and induced downstream transcription without the presence of the trigger sequence, leading to “leakiness”.

This shows the Anderson Series of Promoters with BioBrick name, sequence, and measured strength.


In Lambert iGEM’s 2018 project, the toehold relied on the reporter gene LacZ, a lactose operon encoding the Beta-galactosidase protein (BBa_I732005). When this protein is expressed, it breaks down X-gal into galactose to produce blue pigmentation.

Promoter BBa_J23106 had a measured strength of 0.47, making it a medium strength-promoter, which was optimal for the constraints of the project. The promoter would potentially induce eGFP expression and allow the toehold mechanism to be successful in a dual plasmid transformation with its complementary trigger (BBa_K2550001), while preventing overexpression of the reporter.

Using SnapGene software, promoter BBa_J23106 was inserted in the T7 Promoter Toehold Ribosome Switch with LacZ expression (BBa_K2550000), replacing the BBa_J23100 promoter. In addition, the LacZ Reporter Gene (BBa_K2550201) was identified, deleted, and replaced with eGFP (BBa_E0040) obtained from the iGEM parts registry to create the new parts.

Results

Analysis of Results

To address the problem with overexpression in BBa_K2550000 T7 Toehold, Lambert iGEM used SnapGene software to build two Toehold eGFP constructs with strong promoter BBa_J23100 and medium-strength promoter BBa_J23106. Last year, part BBa_K2550000 was induced by promoter BBa_J23100 and expressed significant leakiness. The team cloned and transformed these two new toehold constructs with the medium-copy plasmid, pSB3C5, in Dh5-alpha E.coli cells. The trigger sequence in pSB6A1 complementary to the De-Novo Toehold sequence used in the proof of concept was isolated from a glycerol stock created by Janet Standeven in conjunction with the Styczynski Lab at the Georgia Institute of Technology. The pSB3C5 toehold-containing plasmids were further transformed with the complementary pSB6A1 trigger-containing plasmid in a dual plasmid transformation to initiate the toehold mechanism and express eGFP.

BBa_K2974316

The transformation of the team’s composite part BBa_K2974316, the BBa_J23106 (medium-strength) promoter with the Toehold eGFP construct, with pSB3C5, did not produce green fluorescent colonies. The absence of fluorescence was ideal, as the reporter protein eGFP should only be expressed in the presence of the trigger sequence. The red fluorescent colonies seen in both images is a result of the backbone, pSB3C5, which was originally cloned with Red Fluorescent Protein (RFP). Lambert iGEM obtained this stock of pSB3C5 from the iGEM Distribution Kit and proceeded to digest the plasmid with High Fidelity EcoRI and PstI restriction enzymes the red fluorescence observed on each plate indicates that the plasmid re-ligated to the RFP insert originally present in the pSB3C5 stock instead of the toehold construct during ligation with T4 ligase. The team inoculated colonies that were not producing RFP which appeared translucent after a 24 hour incubation period. Sequencing then confirmed that colony 3 included the correct toehold sequence.

This shows the 1% gel with the 2-log ladder in well 5 and colony 3 in well 6. Both the insert (toehold - approximately 900 base pairs) and the vector (pSB3C5 - approximately 3000 base pairs) are present in colony 3. When this colony was sent out for sequencing, it aligned with the desired toehold sequence.

This is a picture of the sequencing results from IDT confirms that the toehold construct assembled with pSB3C5 aligns with the desired toehold sequence.

Dual Plasmid Transformation of BBa_K2974101 and BBa_K2550001

The dual plasmid transformation of the part BBa_K2974101, in pSB3C5, and part BBa_K2550001, the trigger sequence, in pSB6A1, produced green fluorescent colonies. The team decided to plate the transformations on two plates with different antibiotics - chloramphenicol/ampicillin and chloramphenicol/carbenicillin - as plasmid pSB6A1 allows for transformed E. coli cells to gain both ampicillin and carbenicillin antibiotic-resistance. Both of the dual plasmid transformations produced low-grade green fluorescence in colonies, which indicated a successful binding of the toehold and trigger sequence and expression of eGFP. There was a 48 hour incubation period required to produce fluorescence.

This shows a picture of the dual-plasmid transformations of the BBa_J23106 Toehold construct (plate 1 containing carbenicillin/chloramphenicol on the left and plate 2 containing ampicillin/chloramphenicol on the right) of the toehold assembled with pSB3C5 and the trigger assembled with pSB6A1. A successful binding of the toehold and the trigger shows low-grade fluorescence from the expression of eGFP.

BBa_K2974700

The transformation of the team’s composite part BBa_K2974700, BBa_J23100 (strong) promoter and Toehold eGFP construct, in pSB3C5, produced both translucent and red pigmented and fluorescing colonies. The red fluorescent colonies seen in the image is a result of the backbone, pSB3C5, which was obtained from the iGEM Distribution Kit and was originally cloned with RFP, re-ligating with its original insert rather than the toehold. The team inoculated the colonies that were not producing RFP. Sequencing then confirmed that colony 3 included the correct toehold sequence.

Conclusion

Reflection

Part BBa_K2550000, contributed by the 2018 iGEM team, consisted of a T7 promoter Toehold Ribosome Switch with LacZ expression. The team looked to improve upon the overexpression of the reporter gene when not induced by the corresponding trigger sequence, BBa_K2550001. The team began by modeling and testing the effect of changing Promoter sequences and Ribosomal Binding Sites on a similar construct using Biobuilder’s BBa_J10050- BBa_J10058 Promoter and RBS combinations.

The T7 promoter constructed with the 2018 toehold was BBa_J23100, which had a reported strength of 1, making it the strongest promoter of the Anderson Series of Promoters barring the consensus strain. Through research, modeling and experimentation, the team identified promoter BBa_J23106 as an optimal promoter for the LABYRINTH project. Promoter BBa_J23106 had a measured strength of 0.47 on a scale of 0-1, making it a medium-strength promoter. This information led us to change the promoter in BBa_K2550000 from BBa_J23100 to BBa_J23106.

In order to characterize the difference in expression of the target reporter gene between promoters BBa_J23100 and BBa_J23106, the team constructed two toehold switches with the reporter protein eGFP. The proof of concept toehold constructions were modeled after experimental C. elegans toeholds designed by the team, which utilized BBa_J23106 as a promoter and eGFP as a reporter.

The light intensity (lux) values, or quantifiable fluorescence, of liquid cultures of the trigger, toehold, and dual plasmid transformations enabled the team to compare the leakiness of both systems. The difference in lux values and the levels of reporter expression in both systems provided justification for improvement of our system via promoter change.

In order to characterize the difference in expression of the target reporter gene between the two promoters, the team constructed two toehold switches with the reporter protein eGFP. Both the BBa_J23100 Toehold and BBa_J23106 Toehold were synthesized using restriction digest and ligation, and were successfully transformed and sequenced. The light intensity values, or quantifiable fluorescence, of the individual transformations of the toehold and of the dual plasmid transformations of the toeholds and the trigger allowed for comparison of leakiness, overexpression that the team observed when using promoter BBa_J23100 the previous year. There was a four day incubation period required to induce eGFP expression within the BBa_J23106 dual plasmid cells, likely due to the cellular demands of dual plasmid transcription and translation. In contrast, there was only a two-day incubation period required by the strains containing the part BBa_K2974101 assembled with BBa_K2550001, possibly due to the relative strength of the BBa_J23100 promoter over the BBA_J23106 promoter.

Comparison of BBa_J23100 promoter and BBa_J23106 promoter with FluoroCents Data

The team developed an inexpensive and accurate fluorometer, FluoroCents, in order to quantify the relative fluorescence (lux values) of the trigger, toehold, and dual plasmid transformations. This allowed for eGFP expression comparison between promoters BBa_J23100 and BBa_J23106, justifying the team’s promoter change to diminish leakiness. Data from FluoroCents show that plain LB, plain E. coli cells, and E. coli transformed with the trigger produced, on average, an equal amount of fluorescence for both promoter-toehold constructs: approximately 2.4 lux.

The toehold constructed with promoter BBa_J23100 produced some fluorescence, resulting in a lux value of 3.19. This is due to the unintended transcription and expression of eGFP without the initiation of the complementary trigger sequence resulting from the strong promoter. In contrast, the toehold constructed with promoter BBa_J23106 produced no fluorescence with a recorded lux value of 2.5. The data supports the hypothesis that the medium-strength promoter (BBa_J23106) did not cause overexpression of the reporter protein, unlike the strong promoter (BBa_J23100).

The dual plasmid transformation of the toehold constructed with BBa_J23100 and the trigger sequence produced a high level of fluorescence, with a lux value of 4.5. This suggests an overexpression of eGFP when the BBa_J23100 toehold construct and its complementary trigger have successfully binded. In contrast, the dual plasmid transformation of the toehold constructed with BBa_J23106 and the trigger sequence produced a lower level of fluorescence with a lux value of 3.67. This suggests a moderate level of transcription and expression of eGFP when the toehold and trigger have successfully binded resulting from the medium-strength promoter (BBa_J23106).

In order to apply this improvement to part BBa_K2550000, Lambert iGEM re-built the T7 Toehold LacZ construct in SnapGene with part BBa_J23106 as the new promoter. The team then began the cloning workflow using Gibson Assembly, aiming to compare pigmentation of transformed E. coli containing the individual toehold and trigger, as well as a dual plasmid transformation of both.

The improved part is BBa_K2974500 and is currently, as of wiki freeze, in the cloning stage, but the team expects to update the registry with the final results after the 2019 Giant Jamboree.

This shows the toehold LacZ Construct with J23106 promoter designed in SnapGene.


This is the LacZ Toehold Construct with J23100 Promoter designed in SnapGene.


This shows the transformation of the LacZ Toehold with the strong promoter, currently without pigmentation.


This is the transformation of the LacZ Toehold with the medium-strength promoter, currently without pigmentation.


PCR products of LacZ fragments (approximately 3000 base pairs), wells 4, 5, and 7 show faint bands at around 3000 base pairs when referenced to the 2-log ladder in well 6.


REFERENCES

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