Part:BBa_K3301003
EndoA Cell Growth Inhibitor
This part is an improvement on the 2012 HKUST team's part BBa_K733012. Their part has a xylose inducible promoter that expresses an endotoxin in order to inhibit cell growth. According to HKUST, their part seemed to be effective when in a solution of .15% xylose, which they determined to be the minimal effective concentration. However, when we attempted to replicate their data, we were unable to replicate their results. We ran multiple experiments, but each time we got inconsistent data. We attribute this to contamination either while ligating and transforming, or in the distribution kit itself. Our part has two improvements on the original design, both in design theory and actual practicality. HKUST's part has 2 major flaws. The first, and easier to fix, was that they used an overly complex terminator. The added strength was unnecessary, and it was prone to issues during synthesis. The old terminator was replaced with part BBa_B1002. The second change to the initial part came from a reworking of the system. The original design had the toxin released when in the presence of xylose. So, xylose would need to be delivered in the body to kill the cells. We believe that this would more often than not increase the potential for unregulated cell growth. While in the body, a myriad of problems could arise that delay or entirely halt the xylose delivery, resulting in an unchecked system. So, to increase safety, we rearranged the parts so that the toxin would be released when deprived of xylose. This way, we can ensure safety because there is little xylose naturally in the body. In conclusion, this killswitch releases a toxin that cleaves mRNA, leading to stagnated cell division. This killswitch can be combined with another part in order to make the whole system dependent on xylose.
After experimentation with this part, we have concluded that it works as intended. We grew cells for 18 hours at 37C in M9 media supplemented with 0.4% glycerol, 35 ug/ml chloramphenicol and various concentrations of xylose. We then measure the OD at 600 nm.
Our control is a part that is not in any way dependent on xylose and it grows at all xylose concentrations.
The xylose-induced toxin (BBa_K733012) works somewhat and the concentration of bacteria starts to go down above an xylose concentration of 0.2%
We have 3 clones of our construct (BBa K3301003) and they work at different levels. Clone 102 produces low cell growth at all concentrations of xylose and has the strongest effect. Clones 2 has very little effect without xylose (as we expect) and then the cells start to grow once xylose is present.
Clone 1 has lower growth at 0% xylose compared to with xylose, but it has a smaller effect than the other two. We will need to figure out why these three clones respond to different levels of xylose.
But for all three, there is an immediate jump as the xylose amount increases. This is to be expected, as the increased xylose concentration means that the inducible promoter is expressing antitoxin so that the cells can live. The part is therefore working as expected.
We also grew each of the parts with glucose (in LB media). All constructs grew almost equally in LB media. This means that none of the parts made the cells sick and the low growth in 0% xylose was due to the xylose concentration and not another reason.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1341
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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