Results

a)

For EryK

(BBa_K4447001)

The first induction was performed following our induction protocol, with a temperature of 37 °C for 4 hours after reaching OD greater than 0.4 and adding arabinose.

Figure 1: Each sample corresponds to a colony, where the sample number corresponds to the colony number, plus a negative control. Each lane was loaded with 20 uL, 10 uL of supernatant and 10 uL loading buffer. The ladder used was: PageRuler Plus Prestained Protein Ladder. The red rectangle shows the location of our EryK protein with a weight of 43.5 kDa.

In these gels, ten samples and a control were run, where each sample corresponds to a different colony. To avoid the use of a possible cheater in addition to evaluate and determine the best colony, a densitometric analysis was performed to determine the concentration of the protein by comparing it with the intensity of a band of the ladder that has an already defined concentration.

Table 1: Estimated concentration of each sample. this was processed thanks to ImageJ (NIH, USA)

As we can see in the results of the densitometric analysis, it resulted in a null expression of our protein both in the control and in colony number 10, telling us that it is possibly cheater and that our induction protocol is successful, because of the way they were expressed in the other colonies and in the negative control the expected results were obtained.

b)

For FRET System

(BBa_K4447004)

The second experiment was performed following our induction protocol, with a temperature of 37 °C until it reach OD greater than 0.4, and after adding arabinose, we let bacterias growth overnight at 25 °C.

Figure 2: Each sample corresponds to a colony, where the sample number corresponds to the colony number. Each lane was loaded with 20 uL, 10 uL of supernatant and 10 uL loading buffer. M: PageRuler Plus Prestained Protein Ladder. C1 and C2: controls not induced with L-arabinose.

In our gel we did not see conclusive results, we did not see evidence of our enzyme in the supernatant, so we can analyze different possibilities, that the expression at 25 °C was not adequate and as the enzyme is quite large a more accelerated cellular machinery is necessary, another option is the formation of inclusion bodies, protein aggregates, which are formed by a recombinant protein, being not native to the species and being of a fairly large size tends to precipitate. The other option is that we find cheaters or self-ligating colonies.

COLLABORATION

Tec-Chihuahua and our team organized a collaboration to validate the enzyme activity of our BioBrick BBa_K4447001. To carry out these experiments, we cloned this part into pBAD/Myc-HisB and sent it to Tec-Chihuahua. Afterward, we requested them to induce protein overexpression at 16 °C and 0.2% arabinose, assuming slower cellular metabolism would reduce protein misfolding. After induction, Tec-Chihuahua ran an SDS-PAGE.

Figure 3. SDS-PAGE results of the EryK protein. M: Molecular mass marker (Thermo Scientific, Waltham, MA, USA)

As shown in

Figure 3

, no sign of our protein was seen, so we can conclude that these conditions were not favorable for producing our protein. For instance, we opted to change our protein overexpression conditions to 25 °C but with the same arabinose concentration.

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