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
- Get PARTS:pSB1A2, pSB3K3, pSB1AK3, R0010, R0040(from BioBricks)
- Dissolution with 15ul d[math]\displaystyle{ H_2O }[/math];1ul for transformation ,14ul stored in -20℃.
- <Transformation STANDARD PROTOCOL>
- Screen positive colonies from plate ,Culture in liquid LB and mini-prep, stored in -20℃
Step 1:taRNA & crRNA synthesize
- 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 .
- Order the primers from TaKaRa.
- 5 cycle PCR to synthesize dsDNA.
- Optional:Enzymatic Digestion with the proper enzyme and cloning into vector (pSB1A2).
- For some taRNAs, assemble them into double taRNAs with Standard Method ("2 primer strategy"').;
Step 2:E0040 -> pSB3K3
- Get E0040,pSB3K3 plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors pSB3K3:EcoRI/XbaI. Fragment E0040:EcoRI/SpeI (0.5uL for each enzyme)
- <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- Update Product State on WIKI
Step 3:E0040(pSB3K3) <- B0015
- Get B0015,E0040(pSB3K3) plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors E0040(pSB3K3):SpeI/PstI. Fragment B0015:XbaI/PstI (0.5uL for each enzyme)
- <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- 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
- Get R0040 plasmid from refrigerator, crRNA from Step1 PCR.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors R0040:SpeI/PstI. Fragment crRNA: XbaI/PstI (0.5uL for each enzyme)
- <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- Update Product State on WIKI
- Sequencing
Step 5:Step4 -> Step3
- Get Step3,Step2 positive plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors Step3: EcoRI/XbaI,fragment Step4:EcoRI/SpeI (0.5uL for each enzyme)
- <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- Update Product State on WIKI
- Sequencing
Step 6:R0010 <- taRNA
- Get R0010,taRNA positive plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors R0010: SpeI/PstI,fragment taRNA:XbaI/PstI (0.5uL for each enzyme)
- <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- update Product State on WIKI
- Sequencing
Step 7:Step5 <- B0015
- Get Step5,B0015 plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors Step6:SpeI/PstI. Fragment B0015:XbaI/PstI.(0.5uL for each enzyme)
- <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- Step6 competent cell.
- Update Product State on WIKI
*Note: Step 2,3, Step 4 and Step 6,7 can be parallel.
Step 8: Step5 and Step7 Cotransformation
- Get step4 positive plasmid and step5 competent cell from refrigerator.
- transform step4 plasmid into step5 cells. <transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB.
Step 9: Induction and Measure the Fluorescence Intensity
- Culture until OD = 0.4 ,diluted to 1:1000.
- 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.;
- Incubate at 28℃
- <Measure the Fluorescence Intensity STANDARD PROTOCOL>