Team:William and Mary/3G Mixed
3G Parallel Circuit Testing
As discussed in the overview, 3G assembly allows for easy assembly of circuits within a single day. Moreover, thanks to the modularity of 3G assembly, it can also be used to simultaneously create multiple variants of the same circuit at once.
This is accomplished by simply putting in a mixture of the part(s) that you want to modify. For example, say you wanted to create variants of a circuit that constitutively express GFP at different levels of brightness. In order to accomplish this, the first thing you would want to do is to change the promoter. With 3G assembly, you can follow the protocol as normal, adding all of the parts to the Golden Gate reaction, but instead of adding 0.5uL of one promoter, you can make a mixture of multiple promoters (say a 3uL mixture with .5uL of 4 different promoters). You can then take 0.5uL of this mixture, put it in the Golden Gate tube, and run the rest of 3G assembly as normal.
This gives each circuit an approximate one in four chance of having any given promoter. With this method, you should observe four different brightness levels in your bacteria once they are transformed. The complexity of parallel circuit creation can be increased if you choose to vary more parts. As long as each circuit ends up with a promoter, RBS, coding sequence, and terminator, a nearly endless of circuit varieties can be assembled and screened.
In our project, we used parallel 3G circuit creation to build multiple variants of a circuit we hoped would function as a temperature inducible IFFL. To do this we assembled three different transcriptional units as follows:
Transcriptional Unit 1
| 0.5µL | WM18_AB_007 |
| 0.5µL | WM18_AB_010 |
| 0.5µL | WM18_CD_004 |
| 0.5µL | WM18_CD_006 |
| 0.5µL | Promoter Mixture |
| 0.5µL | WM18_BC_006 |
| 0.5µL | Coding Sequence Mixture |
| 0.5µL | WM18_DE_001 |
| 0.5µL | 50nM UNS1 A |
| 0.5µL | 50nM UNS3 E |
| 2.0µL | Mastermix |
Transcriptional Unit 2
| 0.5µL | WM18_AB_007 |
| 0.5µL | WM18_AB_010 |
| 0.5µL | WM18_BC_001 |
| 0.5µL | WM18_BC_002 |
| 0.5µL | WM18_BC_003 |
| 0.5µL | Promoter Mixture |
| 0.5µL | 5' Untranslated Region Mixture |
| 0.5µL | WM18_CD_002 |
| 0.5µL | WM18_DE_002 |
| 0.5µL | 50nM UNS3 A |
| 0.5µL | 50nM UNS4 E |
| 2.0µL | Mastermix |
Transcriptional Unit 3
| 0.5µL | WM18_AB_001 |
| 0.5µL | WM18_AB_002 |
| 0.5µL | WM18_AB_006 |
| 0.5µL | WM18_CD_010 |
| 0.5µL | WM18_CD_012 |
| 0.5µL | Promoter Mixture |
| 0.5µL | WM18_BC_005 |
| 0.5µL | Coding Sequence Mixture |
| 0.5µL | WM18_DE_003 |
| 0.5µL | 50nM UNS4 A |
| 0.5µL | 50nM UNS10 E |
| 2.0µL | Mastermix |
These units contain mixtures of many different types of parts. With all of the potential circuit combinations used, we screened for a pulse (the desired output) on our plate reader. However, after analyzing our data we found no such pulses. What we did find was three general cases:
Figure 1: In this case, the fluorescence of the cells was far too low. It appears mf-Lon activity was too strong given the amount of mScarlet being produced.
Figure 2: In this case, it appears that mf-Lon activity is too weak for the amount of mscarlet being produced, causing fluorescence to be much too high.
Figure 3: In this case, the induction of the circuit caused cell growth to radically slow down. For this particular example, the cells took nearly 24 hours to double the OD600.
As a result, none of the cases produced promising IFFL pulses. While unfortunate that no pulses were observed, this experiment was quite valuable in that it showed no possible combination of parts could result in IFFL behavior with the ts-CI system. This is most likely due to the very narrow range of induction parameters provided by ts-CI, as this system relies upon an extremely strong phage promoter. Therefore, it was understood that a different heat inducible system for gene control would be necessary moving forward. Thanks to the power of 3G, we were able to come to this conclusion fairly quickly, as opposed to spending extensive time trying to create and test slight variations on the same circuit. The full raw data for this experiment can be seen on our experiments page.