Part:BBa_K1442107
C2HP Testing Module (reverse GFP)
This is a dedicated testing module containing the C2HP Terminator Part designed to perform characterisation of the RNA Promoters and Terminators in e-coli. A diagram depicting the construct is shown below. The system is designed to work in conjunction with a separate plasmid containing the RdRP coding sequence, called the RdRP Module. As the main distinctive feature of RdRP is its ability to replicate a RNA strand starting from the 3’ end, a coding sequence put in reverse in DNA form will only be expressed if the RdRP is actually performing RNA replication. This is a common set up in experiments designed to study viral replicons and replication processes.
Experimental Design
Test: GFP expression rate in cells co-transformed with plasmid that contains the promoter and a reversed GFP sequence and a plasmid containing the RdRP (Part:BBa_K1442100).
Positive Control: GFP expression rate in cells transformed with Biobrick plasmid that contains a gene for GFP with an inducible T7 promoter.
Negative Control: GFP expression rate in cells co-transformed with the Promoter Testing Module and a plasmid containing a mutated version of the RdRP (Part:BBa_K1442101) that has been proven to be unable to direct successful replication of RNA.
Method
The characterisation of the parts was done using a 96-wells plate fluorescence reader – Tecan.
1. BL21 strain line cells were used to successfully co-transform the two plasmids containing the testing modules shown above. BL21 do not have any pre-existing antibiotic resistance and express the T7 polymerase gene. As many as 12 colonies which have both plasmids were overnighted in 5ml of LB containing the 2 antibiotics ensuring selectivity to make biological replicates. The RdRP plasmid has ampicillin resistance and the Promoters plasmid is resistant to chloramphenicol.
2. The following morning, 10 µl of the overnights were “refreshed” in 1 ml M9 media which unlike LB is not fluorescent but is less rich in nutrients. Again antibiotics were added accordingly.
3. Since the T7 promoter which governs the transcription of both plasmids is IPTG inducible, IPTG of 1mM concentration was added to the refreshed cell cultures.
4. The cells were left to grow and adjust to the new media for 6 hours.
5. The cells were re-refreshed when transferred to the 96 wells plate to be used in the Tecan. 10 µl of cells were added to 190 µl of M9 media plus ampicillin and chloramphenicol antibiotics and 1mM IPTG concentration. Two technical replicas for each biological were made. The experiment was conducted over 21 hours. The data obtained was a numerical value of growth by measuring optical density at 600nm (OD 600) and green fluorescent protein (GFP) taken at different wavelengths. To determine a value for the promoter strength, we averaged the GFP and OD values for all replicas. The GFP was then divided by the OD value to account for difference in growth across the samples due to the effect of the number of plasmids and IPTG.
RdRP-directed Replication
1. The DNA strand gets transcribed and then translated, whereby the RdRP protein is produced.
2. The active RdRP binds to the 3’ end of the transcribed plus –sense RNA strand. It produces its complementary minus-sense RNA strand.
3. The RdRP interacts with the 5’ end of the replicated minus-sense RNA strand. It produces the plus-sense RNA strand again and completes the full replication of the RNA. The new RNA strand can act as a template as well and the process of replication can start over.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 773
Illegal NgoMIV site found at 802 - 1000COMPATIBLE WITH RFC[1000]
| n/a | 3UTR (C2) with hairpin and reverse GFP |