Composite PARTS FOR THE MODULAR, CELL-FREE TRANSCRIPTION ONLY BIOSENSOR.
Our iGEM team has designed multiple composite parts to choose the best working constructs with the best fluorescence to facilitate results visualisation by the naked eye. Firstly, we managed to test in vitro the fluorescence of the RNA aptamer iSpinach (
BBa_K3380150 ) flanked by the F30 tRNA scaffolds (
BBa_K3380101 and
BBa_K3380102 ) using a previously described class III strong T7 promoter (
BBa_Z0251 ) by assembling construct
BBa_K3380500 (results can be seen on the iGEM registry). Similarly, different RNA aptamers can be tested to assess their fluorescence in a cell-free extract. Therefore, we designed the constructs BBa_K3380501-BBa_K3380511 for further characterization of the fluorescent RNA aptamers. This range comprises a combination of different RNA aptamers, presence, or absence of tRNA scaffolds and presence or absence of T7 terminators. Further this list could be expanded with combinations of different T7 promoters, T7 terminators, various tRNA scaffolds and fluorescent RNA aptamers. This would allow the biosensor users to choose the best fluorescent RNA aptamers, based on their performance under the desired conditions.
We managed to test an Arsenic (As) sensing construct (
BBa_K3380600 and
BBa_K3380603 ), using a promoter (
BBa_K3380103 and
BBa_K190015 respectively) that performs transcription upon binding of As atoms. The presence or absence of As in the cell-extract is signalled by the iSpinach fluorescent RNA aptamer flanked by F30 tRNA scaffold. Similarly to the RNA aptamer constructs, we designed composite parts BBa_K3380600-BBa_K3380623 comprising a combination of promoters with As transcription factor binding sites preceding and succeeding the T7 promoter, different RNA aptamers, presence or absence of tRNA scaffolds and presence or absence of T7 terminators. These constructs could be tested to assess the best combination to serve as a test for As identification. Also, several other constructs (
BBa_K3380660 ,
BBa_K3380690 ,
BBa_K3380720 ,
BBa_K3380750 ,
BBa_K3380780 and
BBa_K3380810 ) were designed to be able to signal different heavy metals such as Zn, Pb, HG, Cu, Zn (using
Cupriavidus Metallidurans cell-free extract) , and Co and Ni respectively using transcription factors.
BBa_K3380630 was designed to identify the presence of Fluoride using a transcriptional riboswitch. All the above described composite constructs could be further characterized by using a combination of various fluorescent RNAs, tRNA scaffolds, promoters, and terminators. The table below summarizes all the designed constructs.
| | | Name | Type | Description | Designer | Length |
|---|
| | | W | BBa_K3380500 | RNA | iSpinach fluorescent RNA aptamer construct under T7 RNA polymerase promoter (BBa_z0251) | Alexandru Popov | 183 |
| | | W | BBa_K3380600 | Measurement | TXO ArsR regulated construct under T7 promoter (BBa_K3380103) expressing iSpinach | Alexandru Popov | 218 |
| | | W | BBa_K3380603 | Measurement | TXO ArsR regulated construct under T7 promoter (BBa_K190015) expressing iSpinach | Alexandru Popov | 220 |
| | | BBa_K3380151 | Reporter | DIR2s-Apt fluorescent RNA aptamer | Alexandru Popov | 57 |
| | | BBa_K3380501 | RNA | Broccoli fluorescent RNA aptamer construct under T7 RNA polymerase promoter (BBa_z0251) | Alexandru Popov | 154 |
| | | BBa_K3380502 | RNA | DIR2s-Apt fluorescent RNA aptamer construct under T7 RNA polymerase promoter (BBa_z0251) | Alexandru Popov | 162 |
| | | BBa_K3380503 | RNA | iSpinach fluorescent RNA aptamer construct under T7 RNA polymerase promoter with no scaffold | Alexandru Popov | 117 |
| | | BBa_K3380504 | RNA | Broccoli fluorescent RNA aptamer construct under T7 RNA polymerase promoter with no scaffold | Alexandru Popov | 88 |
| | | BBa_K3380505 | RNA | DIR2s-Apt fluorescent RNA aptamer construct under T7 RNA polymerase promoter with no scaffold | Alexandru Popov | 96 |
| | | BBa_K3380506 | RNA | iSpinach aptamer under T7RNA polymerase promoter (BBa_z0251) and T7 terminator (BBa_K3137010) | Alexandru Popov | 235 |
| | | BBa_K3380507 | RNA | Broccoli aptamer under T7RNA polymerase promoter (BBa_z0251) and T7 terminator (BBa_K3137010) | Alexandru Popov | 206 |
| | | BBa_K3380508 | RNA | DIR2s-Apt aptamer under T7RNA polymerase promoter (BBa_z0251) and T7 terminator (BBa_K3137010) | Alexandru Popov | 214 |
| | | BBa_K3380509 | RNA | No scaffold iSpinach aptamer under T7RNAP promoter (BBa_z0251) and T7 terminator (BBa_K3137010) | Alexandru Popov | 169 |
| | | BBa_K3380510 | RNA | No scaffold Broccoli aptamer under T7RNAP promoter (BBa_z0251) and T7 terminator (BBa_K3137010) | Alexandru Popov | 140 |
| | | BBa_K3380511 | RNA | No scaffold DIR2s-Apt aptamer under T7RNAP promoter (BBa_z0251) and T7 terminator (BBa_K3137010) | Alexandru Popov | 148 |
| | | BBa_K3380601 | Measurement | TXO ArsR regulated construct under T7 promoter (BBa_K3380103) expressing Broccoli | Alexandru Popov | 189 |
| | | BBa_K3380602 | Measurement | TXO ArsR regulated construct under T7 promoter (BBa_K3380103) expressing DIR2s-Apt | Alexandru Popov | 197 |
| | | BBa_K3380604 | Measurement | TXO ArsR regulated construct under T7 promoter (BBa_K190015) expressing Broccoli | Alexandru Popov | 191 |
| | | BBa_K3380605 | Measurement | TXO ArsR regulated construct under T7 promoter (BBa_K190015) expressing DIR2s-Apt | Alexandru Popov | 199 |
| | | BBa_K3380606 | Measurement | No scaffold TXO ArsR regulated construct under T7 promoter (BBa_K3380103) expressing iSpinach | Alexandru Popov | 152 |
| | | BBa_K3380607 | Measurement | No scaffold TXO ArsR regulated construct under T7 promoter (BBa_K3380103) expressing Broccoli | Alexandru Popov | 123 |
| | | BBa_K3380608 | Measurement | No scaffold TXO ArsR regulated construct under T7 promoter (BBa_K3380103) expressing DIR2s-Apt | Alexandru Popov | 131 |
| | | BBa_K3380609 | Measurement | No scaffold TXO ArsR regulated construct under T7 promoter (BBa_K190015) expressing iSpinach | Alexandru Popov | 154 |
| | | BBa_K3380610 | Measurement | No scaffold TXO ArsR regulated construct under T7 promoter (BBa_K190015) expressing Broccoli | Alexandru Popov | 125 |
| | | BBa_K3380611 | Measurement | No scaffold TXO ArsR regulated construct under T7 promoter (BBa_K190015) expressing DIR2s-Apt | Alexandru Popov | 133 |
| | | BBa_K3380612 | Measurement | TXO ArsR regulated construct under T7 P(BBa_K3380103) and (BBa_K3137010) T expressing iSpinach | Alexandru Popov | 270 |
| | | BBa_K3380613 | Measurement | TXO ArsR regulated construct under T7 P(BBa_K3380103) and (BBa_K3137010) T expressing Broccoli | Alexandru Popov | 241 |
| | | BBa_K3380614 | Measurement | TXO ArsR regulated construct under T7 P(BBa_K3380103) and (BBa_K3137010) T expressing DIR2s-Apt | Alexandru Popov | 249 |
| | | BBa_K3380615 | Measurement | TXO ArsR regulated construct under T7 P(BBa_K190015) and (BBa_K3137010) T expressing iSpinach | Alexandru Popov | 272 |
| | | BBa_K3380616 | Measurement | TXO ArsR regulated construct under T7 P(BBa_K190015) and (BBa_K3137010) T expressing Broccoli | Alexandru Popov | 243 |
| | | BBa_K3380617 | Measurement | TXO ArsR regulated construct under T7 P(BBa_K190015) and (BBa_K3137010) T expressing DIR2s-Apt | Alexandru Popov | 251 |
| | | BBa_K3380618 | Measurement | No scaffold TXO ArsR regulated iSpinach construct under T7 P(BBa_K3380103) and (BBa_K3137010) T | Alexandru Popov | 204 |
| | | BBa_K3380619 | Measurement | No scaffold TXO ArsR regulated Broccoli construct under T7 P(BBa_K3380103) and (BBa_K3137010) T | Alexandru Popov | 175 |
| | | BBa_K3380620 | Measurement | No scaffold TXO ArsR regulated DIR2s-Apt construct under T7 P(BBa_K3380103) and (BBa_K3137010) T | Alexandru Popov | 183 |
| | | BBa_K3380621 | Measurement | No scaffold TXO ArsR regulated iSpinach construct under T7 P(BBa_K190015) and (BBa_K3137010) T | Alexandru Popov | 206 |
| | | BBa_K3380622 | Measurement | No scaffold TXO ArsR regulated Broccoli construct under T7 P(BBa_K190015) and (BBa_K3137010) T | Alexandru Popov | 177 |
| | | BBa_K3380623 | Measurement | No scaffold TXO ArsR regulated DIR2s-Apt construct under T7 P(BBa_K190015) and (BBa_K3137010) T | Alexandru Popov | 185 |
| | | BBa_K3380630 | Measurement | TXO Fluoride riboswitch regulated iSpinach construct under Anderson Promoter (BBa_J23119) | Alexandru Popov | 285 |
| | | BBa_K3380660 | Measurement | TXO ZntR regulated construct under BBa_K3380105 promoter expressing iSpinach | Alexandru Popov | 221 |
| | | BBa_K3380690 | Measurement | TXO PbrR regulated construct under BBa_K3380106 promoter expressing iSpinach | Alexandru Popov | 206 |
| | | BBa_K3380720 | Measurement | TXO MerR regulated construct under BBa_K3380107 promoter expressing iSpinach | Alexandru Popov | 208 |
| | | BBa_K3380750 | Measurement | TXO copAB regulated construct under BBa_K3380108 promoter expressing iSpinach | Alexandru Popov | 193 |
| | | BBa_K3380780 | Measurement | TXO czcN regulated construct under BBa_K3380109 promoter expressing iSpinach | Alexandru Popov | 226 |
| | | BBa_K3380810 | Measurement | TXO cnrH regulated construct under BBa_K3380110 promoter expressing iSpinach | Alexandru Popov | 203 |
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- Date: January 2020
- Client: Threads
- Category: Illustration
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- Client: Explore
- Category: Graphic Design
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- Date: January 2020
- Client: Finish
- Category: Identity
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- Client: Lines
- Category: Branding
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- Date: January 2020
- Client: Southwest
- Category: Website Design
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- Date: January 2020
- Client: Window
- Category: Photography
