Auxin Biosynthesis

In the biosynthesis of auxin (IAA), the goal of this experiment is to demonstrate that our bacterium can produce auxin, IAA in particular, for algal development after the transformation of the plasmid containing the three auxin genes (Aro8, Ald-H, and KDC). After transformation, we checked the correct assembly of the plasmid (BBa_K4123001) by performing a diagnostic digest, and the results indicate that the plasmids were assembled correctly with 3 fragments as they gave the correct sizes. After checking the correctness of the plasmid assembly, we carried on checking if the bacteria with the plasmid are able to produce auxin (IAA). By using L-tryptophan as a limiting factor, we compared the wavelength of peaks between our 6 Golden Gate samples (G1-G6) and the control using Ultra Performance Liquid Chromatography (UPLC). Since we aim to detect IAA, the samples were focused on 280 nm which corresponds to IAA. We also ran the pure sample of both IAA (Figure 1) and L-Tryptophan (Figure 2) under the same settings, and the intact vector (Figure 3) was included as a control. Only 3 of the 6 samples showed differences with the vector control, while the other 3 samples showed a similar pattern with the vector control. Moreover, the peaks were not in perfect overlap with IAA (Figure 4). Therefore, the results are inconclusive to show that our bacteria successfully produced IAA after the addition of L-tryptophan. In this case, we were considering additional tests to get more information from the experiment. 

Figure 1: UPLC-DAD chromatogram of IAA at 280 nm. The retention time of IAA is roughly between 0.55 to 0.6 min (0.563 in specific).

Figure 2: UPLC-DAD chromatogram of L-Tryptophan at 280 nm. The retention time of L-Tryptophan is roughly between 0.3 to 0.35 min (0.313 min in specific).

Figure 3: UPLC-DAD chromatogram of the vector (pBbE8c-RFP) at 280 nm. The peaks were roughly between 0.25 to 0.3 min (0.258 in specific) and between 0.8 to 0.85 (0.820 in specific).

Figure 4: UPLC-DAD chromatogram of the 6 samples (G1-G6) at 280 nm.

For more details, please go to the engineering page and the lab book.

Phosphate Uptake

The two transformed bacterial colonies (Transformant A and Transformant B) were grown overnight alongside two controls: a colony composed by the pdCas9-sgRNA-RFP-containing bacteria and a colony containing commercially-competent bacteria. These were grown overnight in M9 minimal medium alongside the inducers arabinose and anhydrotetracycline (which were expected to activate the expression of the dCas9 and the PhoU-gRNA genes). The next morning, after calibrating our spectrometer using the vanadate-molybdate method and a set of phosphate dilutions, the growth medium from these bacterial cultures was extracted and compared. This was done through a set of serial dilutions to increase the sensitivity of the vanadate-molybdate reagent. The reading at 470 nm led to the conclusion that the bacterial cultures had roughly the same phosphate-sequestration capacity, as the absorbance levels presented by the spectrometer were highly similar (table 1). Therefore, we conclude that the experimental results did not provide evidence of the successful engineering of bacteria with a higher capacity to absorb and accumulate phosphate.

Table 1: Table presenting the absorbance values of each one of the serial dilutions of the four growth mediums. It can be observed how the phosphate concentration levels are highly similar between the four mediums, leading to the conclusion that our engineered bacteria did not have a higher capacity to sequester phosphate from the medium.

For more details, please go to our lab book.

Light Switch

The goal of this experiment was to determine whether our Opto-Cre-Vvd recombinase is able to correctly rearrange the genes within our FLEx cassete. To start, we first amplified our LoxP gBlock containing our FLEx cassette using PCR. Following the PCR of the LoxP gBlock, a clear band at the desired size of 1.7 kb was obtained.

Figure 5: Gel from our PCR amplification of the gBlock. Lanes 6 and 8 show a band of 1.7kb, indicating the presence of the LoxP gBlock amplification

We then conducted a gel extract of this PCR product, followed by a Gibson assembly with the vector backbone. The Gibson assembly product was then transformed into a fresh culture of competent cells, with both DH5a and Stellar competent cells being used. A number of colonies were formed, which we then used to grow overnight cultures to amplify the Gibson product. Images of the colonies formed are shown in the figure below.

Figure 6: Colonies formed when the competent cells were transformed with the Gibson product. We observed a lawn of colonies for the Stellar competent cells, and a lower amount of colonies for the DH5a competent cells.

We then attempted to detect the presence of the Gibson product within these bacterial cultures using a fluorescence reading. We attempted to detect the presence of the mKate2 fluorescent protein via this fluorescence. However, only fluorescence levels comparable to blank were detected from all 10 samples, meaning our transformation was not successful. Unfortunately, determination of the erroneous part of the experiment was difficult due to the lack of control for the transformation stage. Therefore, a troubleshooting step was conducted via restriction enzyme digestion to figure out if our Gibson assembly had been successful. To do this, we decided to conduct a restriction digest. Upon restriction enzyme digestion with BbsI and XbaI, plasmid with loxP insert would be cut in two places to form two bands of equal size at around 1.7 kb. On the other hand, recircularized vectors with no insert would be cut to form two distinct bands, at 2.7 kb and 1 kb. Below is the result of the restriction enzyme digestion of the Gibson product transformants.

Figure 7: gel electrophoresis results of restriction digestion of our transformants.

As shown in figure 5, all 10 transformants failed to show a clear band at 1.7 kb, indicating that our transformation was unsuccessful and that our loxP gBlock was not cloned into the vector properly. Thus, as our Gibson cloning was unsuccessful, the bacterial colonies that we grew did not contain the fluorescent proteins of interest. This also explains blank fluorescence levels from plate reading. Unfortunately, we were unable to go back to the first stage of our experiment to try and generate a functional Gibson product due to time constraints, and we were forced to leave the experiment here. Given more time, we would have re-attempted the PCR of the loxP gBlock, followed by the Gibson cloning of this product. This would have hopefully yielded a plasmid that we could then transfect into bacteria, and complete the experiment according to the rest of the wetlab protocol.

For more details, please go to our lab book.

RFP Silencing

Gibson assembly was carried out to inset the gBlock. The transformation was carried out and the results of this step are shown in figure 1. A and B are the experimental assembly pCas9. When 50 μL were plated, this did not produce any colonies. B the pellet was plated, and white colonies were observed, which correlates with what is expected as the insert insertion will disrupt the RFP gene (RFP gene preces make red colonies). C and D are the transformants with the assembly that do not contain the insert. No colonies were observed as suggested. E and F are the transformants with pdCas9-sgRNA-RFP. Red colonies are observed in both cases as expected, because the RFP gene will give a red-pink colour to the colonies. G and H are transformants of the Linearized-PCR pdCas9-sgRNA-RFP. No colonies were observed, as expected.

Figure 8: Transformation results after Gibson assembly. A and B are transformants of the assembly pdCas9, 50 μL and pellet were plated respectively. White colonies are observed only in B. C and D are transformants of the assembly pdCas9 without insert, 50 μL and pellet were plated respectively. No colonies were observed. E and F are transformants of the pdCas9-sgRNA-RFP, 50 μL and pellet were plated respectively. Red colonies are observed in both cases. G and H are transformants of the linearized-PCR pdCas9-sgRNA-RFP, 50 μL and pellet were plated respectively. No colonies were observed.

Note: This experiment was interrupted due to time limitation, However, the assembly was completed. Please direct to the lab book for information about the complete protocol.