Plasmid construction

The Vitreoscilla hemoglobin (VHb) biobricks were ordered from IDT as double-stranded, blunt-ended gBlocks. The first part of the wet lab was cloning our VHb (605 bp) constructs into pSB1C3, as mentioned in the experimental design. After several rounds of trial and error in the cloning process, six VHb constructs were successfully amplified in E. coli DH5⍺. Colony PCR, PCR amplification of purified plasmids with specific primers to the gene target, and plasmid digestions were confirmed through gel electrophoresis, as it is shown by fig. 1.

The left image in fig. 1, the result of a colony PCR, shows that we obtained our inserts in the plasmid around 605 bp. The middle image also proves that the VHb inserts was in the plasmid, at around 605 bp band. However, the digestion of the purified plasmid using NotI, in the right image, should cut the DNA plasmid to bands of 898 and 1780 bp in the picture. In that case, only the K2602013 construct was cut properly.

Further confirmation was accomplished by sending in the DNA plasmids to Eurofins Genomics for the sequencing who confirmed all our six BioBricks; initially 4 and then other 2 biobricks. Therefore we were able to confirm the biobricks through all methods. They are BBa_K2602010, BBa_K2602011, BBa_K2602012, BBa_K2602013, BBa_K2602014, BBa_K2602015, and BBa_K2602016.

Expression

After successful transformations, we performed SDS-PAGE (fig. 2). VHb has a molecular weight of 15.8 kDa, which is expected to be just above the second band of the ladder. The gel shows that all our transformations have a band with the expected molecular weight. However, we cannot confirm that it corresponds to VHb as the negative control also has a band with the same molecular weight. It is possible that there are other proteins expressed in E. coli of the same molecular weight, making the VHb difficult to discern. Due to these inconclusive results, we would have liked to perform a western blot analysis. However this wasn’t possible, as there are no commercially available antibodies for VHb. One way to confirm expression of the protein could be to express it with a His-tag, which would allow for purification, and then perform the SDS-PAGE.

When expressing VHb, we found that addition of Aminolevulinic acid (ALA) increased the red color which is characteristic of hemoglobin (fig. 2). ALA is a precursor to heme, a ligand necessary for the function of hemoglobin. Bubbling the cultivations with carbon monoxide (CO), which is commonly done when expressing human hemoglobins, also increased the red color.

When expressing VHb, we found that addition of Aminolevulinic acid (ALA) increased the red color which is characteristic of hemoglobin (fig. 3). ALA is a precursor to heme, a ligand necessary for the function of hemoglobin. Bubbling the cultivations with carbon monoxide (CO), which is commonly done when expressing human hemoglobins, also increased the red color.

Growth curve analysis

The growth curves (fig. 4) of the VHb composite BioBricks were analyzed to see if VHb expression had any effect on growth rate, final OD or lag time and if the promoter strength influenced this effect. The curves were analyzed through a statistical model-based approach where the Gompertz sigmoid was used to to estimate the growth rate.

Parameter estimates with a 95% confidence interval are shown in (fig. 4). For the final OD600, there was no significant difference between the constructs or compared to the negative control. The lag time or time to exponential growth was also determined to not be significant.

Parameter estimates with a 95% confidence interval are shown in (fig. 5). For the final OD600, there was no significant difference between the constructs or compared to the negative control. The lag time or time to exponential growth was also determined to not be significant.

However, there is a significant difference in growth rates at a level of 90% for all VHb-containing biobricks except K2602011 compared to the negative control. For K2602014 and K2602015, the effect was significant also on a 95% confidence level. Interestingly these two have the lowest promoter strengths, with the relative strengths 0.00 and 0.01, respectively. However, we have results suggesting that the expression level in 2014 is not 0, but that the promoter is in fact leaking to some extent. The implication of this is that a low level of VHb expression has a positive effect on the growth rate.

It was further observed that the increase in growth rate was negatively correlated with increased promoter strength. It seems therefore that only a small amount of VHb is enough to enhance growth rate. The results also indicate that cells expressing VHb grow faster during the exponential phase and therefore reach stationary cell density faster than those without. However, while we were not able show an increased stationary cell density with statistical significance, we still believe the results may be of biological significance. We suggest more studies to be done in order to fully evaluate the effect of VHb. Read more about the results of the growth curve analysis here.

K2602020-K2602026

Plasmid construction

For the proof-of-concept we needed plasmids containing both a VHb biobrick and green fluorescent protein (GFP), for which we used the biobrick BBa_J364000. We tried three methods to assemble the constructs: 3A assembly, Gibson assembly and restriction enzyme cloning. In the end, biobrick BBa_K2602026 was assembled through gibson assembly and biobricks BBa_K2602020, BBa_K2602021, BBa_K2602023, BBa_K2602024 and BBa_K2602025 were assembled through restriction enzyme cloning.

All the parts were confirmed with agarose gel electrophoresis (fig. 6). The size of the VHb-GFP gene is in total around 1.5 kbp. The left picture of fig. 6 shows that the bands of the constructs are found slightly above the fifth ladder from bottom, which is around 1.5 kb. Though the bands are difficult to see in the figure, this was due to quenching caused by the high intensity of the other bands and they could be visualized by hiding those bands. Another confirmation was done by cutting the plasmid with NotI and gave the bands at around 1.5 kbp and 2 kbp. The constructs were also sent for sequencing, however only the construct BBa_K2602021 was confirmed through this method. The likely reason for this is use of sub-optimal sequencing primers. In total, six constructs of VHb-GFP were assembled and transferred into E. coli BL21 for co-expression.

Expression

After successful transformations were confirmed, SDS-PAGE (fig. 7) analysis was performed to confirm the coexpression of both proteins; VHb and GFP. In the figure, from the second column from the left to right it is possible to observe the protein profile of the constructs with the following BioBricks: BBa_K2602020, BBa_K2602021, BBa_K2602023, BBa_K2602024, BBa_K2602025 and BBa_K2602026. VHb has a molecular weight of 15.8 kDa which is expected to be just above the second band of the ladder, while GFP has a molecular weight of 27 kDa which is expected to be just above the fourth band of the ladder. The gel shows that all our transformations have the bands with the expected molecular weights.

It is important to highlight that no negative control was used, as our control strain also has a band with a molecular weight like one of VHb and therefore the confirmation of the expression of VHb should instead be done by its expression with a His-tag, which would allow for purification before performing the SDS-PAGE.

Finally, the expression of the VHb-GFP constructs were also confirmed by a physical inspection of the cell pellets of the constructs at the end of the cultivations. As can be appreciated in , the pellets exhibit a green color that is characteristic of the production of GFP. Therefore is reasonable to declare that all the transformants were able to express GFP.

The transformants were evaluated under different conditions of the headspace to establish the effect of VHb on GFP production under conditions of high and low oxygen transfer rate due to the variation of the surface area of the media. The GFP content of each constructs at different media levels were plotted against the relative strength of the VHb promoter used in the biobrick fig. 9. The plots were linearized with a simple linear regression where their parameters were obtained along with their 95% two-sided confidence intervals

By looking at the intervals of the slope (Table 1) it can be clearly seen that the promoter strength on VHb has a significant positive effect on the level of expression of GFP, especially at low media levels.