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：pSB1A2, pSB3K3, pSB1AK3, R0010, R0040(from BioBricks)
2. Dissolution with 15ul d[math]\displaystyle{ H_2O }[/math]；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. 5 cycle PCR to synthesize dsDNA.
4. Optional:Enzymatic Digestion with the proper enzyme and cloning into vector (pSB1A2).
5. For some taRNAs, assemble them into double taRNAs with Standard Method ("2 primer strategy"').;

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:E0040(pSB3K3) <- B0015

1. Get B0015，E0040(pSB3K3) plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors E0040(pSB3K3)：SpeI/PstI. Fragment B0015：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

*Note: Step 2,3 are only needed in the construction of the first lock-efficiency-test plasmid. The product of Step 2,3 can be stored and remains useful in the following constructions of the other 2 lock-efficiency-test plasmids.

Step 4: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 5:Step4 -> Step3

1. Get Step3，Step2 positive plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors Step3: EcoRI/XbaI，fragment Step4：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 6:R0010 <- taRNA

1. Get R0010，taRNA 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 7:Step5 <- B0015

1. Get Step5，B0015 plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors Step6：SpeI/PstI. Fragment B0015：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. Step6 competent cell.
9. Update Product State on WIKI

*Note: Step 2,3, Step 4 and Step 6,7 can be parallel.

Step 8: Step5 and Step7 Cotransformation

1. Get step4 positive plasmid and step5 competent cell from refrigerator.
2. transform step4 plasmid into step5 cells. <transformation STANDARD PROTOCOL>
3. Screen Positive Colonies from Plate ,Culture in liquid LB.

Step 9: Induction and Measure the Fluorescence Intensity

1. Culture until OD = 0.4 ,diluted to 1:1000.
2. Three Groups are divided;
   1. Group A Control group, with nothing add. ;
   2. Group B Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L);
   3. Group C Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L) and aTc (30ng/mL), induced separately.;
3. Incubate at 28℃
4. <Measure the Fluorescence Intensity STANDARD PROTOCOL>