IGEM:Peking/2007/Count-Procedure-Riboregulator

taRNA&crRNA Testing

Important Note:In assembly step titles, parts names are positioned as in the product vectors (5' part on the left). The A->B and B<-A means inserting A into B, indicating A is the fragment and B is the vector. Thus when using ->, the digestion enzymes will be EcoRI/XbaI for the vector and EcoRI/SpeI for the fragment; when using <-, the digestion enzymes will be SpeI/PstI for the vector and XbaI/PstI for the fragment. Refer to the standard assembly method for explanation.

Step 0: pre-Exp

1. Get PARTS：pSB1A3, pSB3K3, pSB1AK3, R0010(from BioBricks)
2. Dissolution with 15ul dH20,7；1ul for transformation ,14Ul stored in -20℃.
3. <Transformation STANDARD PROTOCOL>
4. Screen positive colonies from plate ,Culture in liquid LB and mini-prep, stored in -20℃

Step 1:taRNA & crRNA synthesize

1. Synthesize taRNA and crRNA (TaKaRa): Parts with BioBrick prefix and suffix flanked. Designing Primers: two primers covering 5' and 3' of the desired fragment respectively .
2. Order the primers from TaKaRa.
3. 1 cycle PCR to synthesize dsDNA.
4. Enzymatic Digestion with the proper enzyme and cloning into vector (pSB1A2).
5. For some taRNAs, assemble them into double taRNAs with Standard Method.;

Step 2:E0040 -> pSB3K3

1. Get E0040，pSB3K3 plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors pSB3K3：EcoRI/XbaI. Fragment E0040：EcoRI/SpeI (0.5uL for each enzyme)
3. <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
4. <DNA ligation STANDARD PROTOCOL>
5. <Transformation STANDARD PROTOCOL>
6. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
7. Test with <restriction enzymes Digestion STANDARD PROTOCOL> ，if Positive , Go next step.
8. Update Product State on WIKI

Step 3:R0040 <- crRNA

1. Get R0040 plasmid from refrigerator, crRNA from Step1 PCR.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors R0040：SpeI/PstI. Fragment crRNA: XbaI/PstI (0.5uL for each enzyme)
3. <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
4. <DNA ligation STANDARD PROTOCOL>
5. <Transformation STANDARD PROTOCOL>
6. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
7. Test with <restriction enzymes Digestion STANDARD PROTOCOL> ，if Positive , Go next step.
8. Update Product State on WIKI
9. Sequencing

Step 4:Step3 -> Step2

1. Get synthesized RNA，Step 2 positive plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors Step2: EcoRI/XbaI，fragment Step3：EcoRI/SpeI (0.5uL for each enzyme)
3. <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
4. <DNA ligation STANDARD PROTOCOL>
5. <Transformation STANDARD PROTOCOL>
6. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
7. Test with <restriction enzymes Digestion STANDARD PROTOCOL> ，if Positive , Go next step.
8. Update Product State on WIKI
9. Sequencing

Step 5:R0010 <- taRNA

1. Get R0010，Step 3 positive plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors R0010: SpeI/PstI，fragment taRNA：XbaI/PstI (0.5uL for each enzyme)
3. <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
4. <DNA ligation STANDARD PROTOCOL>
5. <Transformation STANDARD PROTOCOL>
6. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
7. Test with <restriction enzymes Digestion STANDARD PROTOCOL> ，if Positive , Go next step.
8. update Product State on WIKI
9. Sequencing

Step 6: Step 4 and Step 5 Cotransformation

1. Get Step 4 positive plasmid，Step 5 positive plasmid from refrigerator.
2. <Cotransformation STANDARD PROTOCOL>
3. Screen Positive Colonies from Plate ,Culture in liquid LB.

Step 7: Induction and Measure the Fluorescence Intensity

1. Culture until OD = 0.4 ,diluted to 1:1000.
2. Three Groups are divided;

Group A Control group, with nothing add. ;

Group B Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L);

Group C Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L) and aTc（30ng/mL）,induced separately;

1. incubate at 28℃
2. <Measure the Fluorescence Intensity STANDARD PROTOCOL>