Difference between revisions of "Part:BBa K2996011"

Revision as of 22:07, 21 October 2019

RSRL-eGFP downstream of pTac

This composite part is the core of our pRead plasmid in information storage device, where input signal is recorded as a permanent spacer. An original pRead contains a CRISPR array (We name it RSRL), which consists of 69 bp leader and one spacer flanked by two repeats. Integration cassette is put upstream of the complete coding sequence of an out-of-frame EGFP gene (EGFP +1). Translation of transcripts generated from the tac promoter will stop at the leader, for those two stop codons. The wrong reading frame and existing stop codon together will prevent expression of the EGFP in the original situation.

Figure 1. Schematic representation of pRead before induction.

Upon information input, that is addition of corresponding inducer, spacer adaptation from cas1-cas2 will result in an addition of 61 base pairs, 33bp new spacer and 28bp replicated repeat, into the integration cassette. Stop codon will be moved out of frame, while EGFP in the open reading frame. Thus, successful translation of EGFP is our desired information output signal, observed by eyes and microplate reader.

Figure 1. Schematic representation of pRead after induction.

Experiments and Results

1.Overnight cultures of co-transformants were transferred and incubated at 37℃, 200rpm until cells grew into exponential stage. </br>
2.Samples were then trisected for different induction patterns.</br>
3.Positive samples were induced with both tetracycline and IPTG. Negative samples were induced with tetracycline only. Blank samples were not induced as control group. Samples were then incubated overnight at 20℃ for 16h.
4.OD600 and fluorescence data were acquired from microplate reader, with excitation wavelength at 485nm and emission wavelength at 528nm. OD600 of all samples were adjusted to 0.5 before fluorescence measurement.

Figure 1. Fig. 1 Fluorescence of cells harboring pTrig, pRec and pRead

Cells induced with IPTG and tetracycline showed significant increase in fluorescence value, indicating successful signal input.

Microscope !!!

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 252
Illegal BamHI site found at 978
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 This composite part is the core of our pRead plasmid in information storage device, where input signal is recorded as a permanent spacer.
+An original pRead contains a CRISPR array (We name it RSRL), which consists of 69 bp leader and one spacer flanked by two repeats. Integration cassette is put upstream of the complete coding sequence of an out-of-frame EGFP gene (EGFP +1). Translation of transcripts generated from the tac promoter will stop at the leader, for those two stop codons. The wrong reading frame and existing stop codon together will prevent expression of the EGFP in the original situation.
-The ribosome-binding site and the translation initiation codon of this reading device are indicated. Translation of transcripts generated from the tac promoter will stop at the leader, for those two stop codons. The wrong reading frame and existing stop codon together will insure no expression of the florescent protein in the original situation.
 <center>{{#tag:html|<img style="max-width: 50%" src="https://2019.igem.org/wiki/images/3/34/T--SJTU-BioX-Shanghai--wet_lab-before.png" alt="" />}}</center>
 <center><b>Figure 1.</b> <i>Schematic representation of pRead before induction.</i> </center>
+Upon information input, that is addition of corresponding inducer, spacer adaptation from cas1-cas2 will result in an addition of 61 base pairs, 33bp new spacer and 28bp replicated repeat, into the integration cassette. Stop codon will be moved out of frame, while EGFP in the open reading frame.
+Thus, successful translation of EGFP is our desired information output signal, observed by eyes and microplate reader.
 <center>{{#tag:html|<img style="max-width: 50%" src="https://2019.igem.org/wiki/images/a/ad/T--SJTU-BioX-Shanghai--wet_lab-after.png" alt="" />}}</center>
 <center><b>Figure 1.</b> <i>Schematic representation of pRead after induction.</i> </center>
-Upon information input, that is addition of corresponding inducer, spacer adaptation from cas1-cas2 will result in an addition of 61 base pairs, 33bp new spacer and 28bp replicated repeat, into the integration cassette. Stop codon will be moved out of frame, while EGFP in the open reading frame.
-Thus, successful translation of EGFP is our desired information output signal, observed by eyes and microplate reader.
+===Experiments and Results===
-===Experiment and Result===
 <br>1.Overnight cultures of co-transformants were transferred and incubated at 37℃, 200rpm until cells grew into exponential stage. </br>
 <br>2.Samples were then trisected for different induction patterns.</br>
@@ Line 26: / Line 25: @@
+<center>{{#tag:html|<img style="max-width: 80%" src="https://static.igem.org/mediawiki/parts/0/04/T--SJTU-BioX-Shanghai--result_1.3.png" alt="" />}}</center>
+<center><b>Figure 1.</b> <i>Fig. 1 Fluorescence of cells harboring pTrig, pRec and pRead</i> </center>
+Cells induced with IPTG and tetracycline showed significant increase in fluorescence value, indicating successful signal input.
-<center>{{#tag:html|<img style="max-width: 80%" src="https://static.igem.org/mediawiki/parts/c/ca/T--SJTU-BioX-Shanghai--big_CaCI2.png" alt="" />}}</center>
-<center><b>Figure 1.</b> <i>floresence change</i> </center>
-Cells induced with IPTG and tetracycline showed significant increase in fluorescence value, indicating successful signal input. However, induction of sole tetracycline displayed a similar effect, indicating ineffectiveness of IPTG induction. This is due to high base expression of tac promoter, confirmed by our further experiments.
 Microscope !!!