Team:MIT/Results
Results
Overview
Cloning - Successes and Failures
Transforming B. subtilis - Successes and Failures
Assays - Successes and Failures
Cloning
Successes
We successfully cloned and verified all of our basic parts and composite parts with the exception of pL1-5,6,16 (BBa_K4074028, BBa_K4074029, BBa_K4074039) and the pL2 composite parts that include these pL1’s (BBa_K4074052, BBa_K4074058, BBa_K4074064). To accomplish this, we used Golden Gate Assembly to assemble our parts, transformed them into NEB 5 alpha competent E. coli cells, screened for white colonies, mini-prepped them, and verified their sequence by Sanger sequencing with Genewiz.
We have also successfully cloned four pL2 backbones (BBa_K4074042 through BBa_K4074049) for our composite pL2 parts that can be integrated into the genome of B. subtilis. These backbones were assembled using both traditional restriction digest ligation cloning and Gibson Assembly using the iGEM pSB1C3 plasmid backbone. We have demonstrated that our composite parts with the pSB_AMY backbone and pSB_AZL backbone can be successfully transformed into B. subtilis and integrated in the genome via homologous recombination into the amyE and azlCD loci.
Failures
We failed to clone the genes bcaP (our importer) and bkdB (a subunit of the BCKDH complex) with the constitutive promoter Pveg. However, we did end up successfully cloning bcaP under the control of an inverted Pveg promoter that can be used in our system with Cre recombinase. More information on our attempts to clone these parts may be found on the Engineering page.
Transforming B. subtilis
Successes
We were able to successfully transform all of the pL2 composite parts with the pSB_AMY backbone and pSB_AZL backbone into the genome of B. subtilis.
We tried two methods of linearizing our pL2 composite parts to transform into B. subtilis:
1) Performing PCR on the plasmids with primers that surround the homology arms
2) Digesting the plasmids with I-SceI (Read more about how we designed these parts on the Parts page!)
To achieve this, we tried transforming different concentrations of the PCR and digest product, and found that the PCR product yielded the highest transformation efficiency. As a control, we also transformed gDNA from the Grossman lab that is known to integrate successfully into the genome.
Below are the results of transforming our linearized pL2 composite parts into B. subtilis:
To perform an initial screening of our colonies, we used the amyE starch test (read more about it on the Experiments page) to screen whether or not our constructs were successfully integrated into the amyE locus. Patches from colonies with something inserted within the amyE locus will not be able to hydrolyze starch while patches from colonies with the intact amyE gene will be able to hydrolyze starch and clear a “ring” around it.
The bottom two patches on each plate are from our control strain of JMA222 with functional amyE, while all of the patches above them are from our transformants. Our transformants do not exhibit any clearing around their edges, indicating that there was something successfully inserted into the amyE locus.
Based on these results, we verified the DNA was integrated into the genome via gDNA extraction and PCR amplification that the insert was the right length, in addition to through sequencing of the PCR product.
The last 3 bands correspond to BBa_K4074050 (Constitutive mApple with Kan Selection). The PCR product is around 2.2kb, indicating that it is the right length. Sequencing the PCR products confirmed this.
In order, the lanes read:
Gel 1:
Lane 2: pl2-2
Lane 3 + 4: pL2-3 (PCR)
Lane 5 + 6: pL2-5 (PCR)
Lane 7 + 8: pL2-6 (PCR)
Lane 9 + 10: pL2-7 (PCR)
Lane 11 + 12: pL2-13 (PCR)
Gel 2:
Lane 2 + 3: pL2-6 (Digest)
Lane 4 + 5: pL2-7 (Digest)
We identified at least one colony for each pL2 that had the right length construct and sent those for sequencing. We received confirmation on those colonies and saved them as glycerol stocks so we could run assays on these strains’ abilities to uptake BCAAs.
Failures
Unfortunately, the pL2’s that we constructed using the pSB_BKD backbone were unable to be successfully integrated into the B. subtilis genome.
In order, the lanes read:
Lane 2 - 5:pL2 with AmyE backbones, already verified
Lane 6 + 7: pL2-12 (PCR)
Lane 8 + 9: pL2-12 (Digest)
Lane 10 + 11: pL2-14 (PCR)
Lane 12 + 13: pL2-14 (Digest)
Lane 14: pL2-17 (PCR)
Not pictured: pL2-17 (Digest)
The shortest bands of the PCR products in lanes 6-13 correspond to the empty bkd operon site, indicating that nothing was successfully integrated at the locus. The lanes with a longer band around 2.5 kb were also incorrect. Sequencing these constructs confirmed this hypothesis.
We only tried this transformation once and plan to try again. We transformed these pL2s with the other pL2s containing amyE backbones so the media and conditions of the transformation were consistent. Assuming that our constructs are correct, we hypothesize that overexpression of the bkd operon might be toxic to the cell, or that integrating something into the native promoter affects the overall fitness of the cell. We found it suspicious that there were many colonies that still grew on these mls antibiotic plates, so we definitely plan to repeat the experiment and troubleshoot.
Assays
B. subtilis Growth Curve
We mapped out a growth curve for B. subtilis by culturing a colony in LB and measuring the OD600 every hour or half hour to generate a population growth curve to be used in our model.
We have also successfully cloned four pL2 backbones (BBa_K4074042 through BBa_K4074049) for our composite pL2 parts that can be integrated into the genome of B. subtilis. These backbones were assembled using both traditional restriction digest ligation cloning and Gibson Assembly using the iGEM pSB1C3 plasmid backbone. We have demonstrated that our composite parts with the pSB_AMY backbone and pSB_AZL backbone can be successfully transformed into B. subtilis and integrated in the genome via homologous recombination into the amyE and azlCD loci.
Assay Kit Testing
To determine the sensitivity of our assay kit to BCAAs, we performed a variety of tests in combination.
First, we generated growth curves for each of the three amino acids in singlicate:
In an ideal situation, the amino acid kit would have the same sensitivity to all three BCAAs so that we would be able to test them in combination while still being able to accurately determine BCAA concentration. However, in the above assay, there appeared to be some variation in sensitivity, particularly for isoleucine.
To better elucidate this difference, we then tested the assay in duplicate, on leucine and valine. We also used a larger range of concentrations to test the linear range of the assay:
We found that the linear range of the assay kit extends to almost 500 μM, and that the absorbance levels appear relatively constant for this range between the four samples.
Additionally, we tested that our leucine dilutions were accurate by comparing two standard curves: one with the leucine stock that we made, and one with the leucine standard provided with the amino acid kit:
Next, we tested various combinations of BCAAs at 125 uM. If the assay kit responds to all three amino acids in the same way, then these samples should all have the same absorbance regardless of their composition.
Fortunately, the values were all very similar with the exception of Leu+Val and Ile+Val, which were due to known mistakes in pipetting. As such, we concluded that the assay kit responded in practically the same way to all three amino acids, allowing us to test all three of them together.
Next, we tested BCAA concentrations when diluted with LB instead of water, and found that the background concentration of BCAAs in LB was too high to get any significant data. Therefore, we decided to transition to growing B. subtilis in minimal media:
BCAA Concentration Assay
With this information, we next moved to performing our assay on B. subtilis. Our first assay was to determine the optimal concentration of BCAAs to grow B. subtilis in. To determine this, we grew B. subtilis in minimal media supplemented with varying concentrations of BCAAs until the cells reached 0.5 OD, at which point we assayed the supernatant of the cells to determine the amount of BCAAs remaining.
Time-point Assay
We ran the time-point assay multiple times. The first time was done with a 400 μM starting concentration of BCAAs.
Our data showed that the concentration of BCAAs decreased over time, suggesting that B. subtilis was consuming BCAAs as expected. However, even at starting time, the absorbance was very low. Therefore, we decided to repeat the assay with a starting concentration of 800 μM. We performed this second assay in duplicate and also included a standard curve.
For this assay, the absorbance values were much higher than in the previous assay. While we did expect the absorbance to increase to some degree since we doubled the BCAA concentration, it is surprising that it did so by so much. In addition, our standard curve saturated out at a much lower concentration than expected. It is also unexpected to see that after a certain time period, B. subtilis no longer appears to be consuming BCAAs as the concentration levels out at an absorbance value of about 0.6.
Transformed B. subtilis Time-point Assay
After transforming B. subtilis with our DNA constructs, we ran multiple time-point assays to determine the differences in leucine consumption rate between them. However, for unknown reasons, our absorbance values were 0.2-0.3 across the board for all samples at all time-points, though the data for our leucine standard was as expected. We also recorded the OD values of each set of samples to compare the effect of our constructs on the growth rate of the bacteria.