Overview

Our project had two genetic approaches: the purR Purine Sensor and the Guanine-II Riboswitch. We went through one full engineering cycle and started on a second for both approaches.

Design

The problem with current methods of measuring varroa mite infestation is their qualitative nature. Our method should be more accurate than current methods like the “sugar shake”.

By creating a biosensor that produces an increasing amount of reporter pigment that correlates to the infestation level of the beehive, we can reliably determine infestation levels and intervene accordingly to save the hive.

Pre-existing natural genetic circuits have been studied and published in peer review articles. Some of these such as the tetR gene have been used and studied so extensively to the point that it is a well established tool used by genetic engineers. By combining documented sequences centered around guanine sensing and genetic tools like the tetR system, we can create a biosensor based on the extra guanine in a varroa mite infested beehive.

The CIDAR MoClo Parts Kit has a wide array of established modular components designed to create tailor made expression vectors through Golden Gate cloning. Designed gene blocks need to be compatible with Golden Gate cloning in the MoClo scheme to fit the parts available in the CIDAR MoClo Parts Kit.

Our bacterial strain should be easily manipulated for many iterative genetic experiments and is low maintenance. DH5-α E. coli will suffice for early experimentation.

Software for building gene blocks and testing their cloning digitally is necessary. We will use the Geneious software.

Build

The construction of our build will revolve around Golden Gate cloning in the MoClo scheme.

• Fast and efficient method of cloning that relies on type two restriction sites that are excised after ligation which prevents backwards reactions (Golden Gate cloning).
• Genetic segments for all parts can be obtained from online databases and sequences can be edited to contain Golden Gate cloning restriction and recognition sites.

Two parts related to the purR system were identified from literature. The first is a purR gene sequence derived from E. coli K12 including a Promoter+RBS+CDS to serve as the guanine sensor and PurR repressor protein producer (1). Additionally a promoter sequence regulated by the PurR repressor protein from the same E. coli K12 was also identified (2)(3).

These sequences were then integrated into the MoClo cloning scheme by adding BsaI recognition sites and adding _AD overhangs to the guanine sensor sequence and _AC overhangs to the purR repressor protein regulated sequence. Additionally, seven extra adenine bases were added to each end of both synthetic sequences to increase cleaving efficiency (4).

Additionally, a variety of terminators with a range of strengths were chosen from the CIDAR MoClo Extension, Volume I and modified slightly to have _DF overhangs instead of _DE overhangs.

A number of sequences were taken directly and without modification from the CIDAR MoClo Parts kit or CIDAR MoClo Extension, Volume I. These are parts such as pTet_AB, BCD2_BC, c99m (mScarlet-I), and tetR_CD.

These parts were combined using a BsaI Golden Gate cloning to produce a single expression vector plasmid. This plasmid was transformed into competent DH5-α E. coli cells.

Test

In order to determine if our purR final construct would successfully produce mScarlet protein in response to guanine concentration, we transformed the construct into competent E. coli cells and inoculated different types of media with 3 isolates of the purR final construct. Each isolate was inoculated into LB, LB+1nM guanine, M9, and M9+1nM guanine. Additionally, all of the M9 media included 1% thiamine, as DH5-α is auxotrophic for thiamine.

Learn

Our purR construct produces the mScarlet reporter protein in LB media but not in M9 media or M9 media supplemented with guanine. This could mean a few things:

1. The guanine we are using is not the correct form of guanine needed to trigger the purR guanine sensor and the correct form is found within the more complex LB media.
2. The purR-tetR-mScarlet pathway needs at least trace amounts of some nutrient found in the LB media and absent in the M9 media in order to fully carry out the pathway from sensing the guanine to expressing the mScarlet reporter.
3. The purR pathway could be sensing and reacting to the concentration of a substrate other than guanine that is found in LB media and not the M9 media.

Design

Before completely re-designing the purR construct, we need to carry out further testing to determine what is wrong with the first iteration.

1. To address the first potential issue, we could order different forms of guanine and repeat testing with a different supplemental guanine.
2. To address the second potential issue, we could try:
   • Growing our construct in 5ml of M9 media supplemented with 50ul of LB media.
   • Growing our construct in 5ml of M9 media supplemented with 50ul of LB media and 1mM of guanine.
   • If a clear difference in the saturation of red color in the broth is observed, then the issue is likely that M9 is too minimal of a media to support at least one part of the purR-tetR-mScarlet pathway.
3. To address the third potential issue, we could try a similar experiment to issue 2:
   • Growing our construct in 5.0ml of M9 media supplemented with 1nM of guanine.
   • Growing our construct in 4.995ml of M9 media supplemented with 5ul of LB media and 1nM of guanine.
   • Growing our construct in 4.95ml of M9 media supplemented with 50ul of LB media and 1nM of guanine.
   • Growing our construct in 4.5ml of M9 media supplemented with 500ul of LB media and 1nM of guanine.
   • Growing our construct in 0.5ml of M9 media supplemented with 4.5ml of LB media and 1nM of guanine.
   • Growing our construct in 5.0ml of LB media and 1nM of guanine.
   • If the sensor is sensing something other than guanine, then we would expect to see that the red color of the broth increases in parallel with the increasing concentration of the LB broth.
   • If the sensor is truly sensing guanine, then we would expect to see a large increase in the red color of the broth as soon as it reaches the amount LB needed to activate the part(s) of the purR-tetR-mScarlet pathway.