EXISTING BIOBRICK CHARACTERISATION

As part of the criterion for the Gold medal, we set about trying to improve the characterisation of an existing part on the registy. In line with the rest of our project, we felt it pertinent that we characterise something relevant and as such we chose this part. The information below is taken from the registry page:

Introduction
Warwick iGEM 2014 used this part as a positive control for our experiments investigating promoter strengths for a self replicating RNA strand, using the Hepatitis C Virus NS5B gene encoding RNA dependent RNA polymerase, as the replicating enzyme.

This was measured using a tecan Magellan plate reader in which fluorescence in the GFP range was measured over a period of 24 hours with both biological and technical replicates for each transformed cell type.

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 (Part:BBa_J04430). Promoter induction strength in response to different concentrations of added inducer (IPTG) was characterised.
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
This was measured using a Tecan Magellan plate reader in which fluorescence in the GFP range was measured over a period of 24 hours with both biological and technical replicates for each transformed cell type.
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.

Results
The fluorescence and OD of the cells was measured over 20 hours and at 4 different amounts of IPTG; 0μl, 4μl, 20μl, 100μl.

Graph showing fluorescence over time in in BL21 cells transformed with the IPTG inducible GFP plasmid and different amounts of IPTG including error bars in each case. This graph demonstrates that 4μl IPTG displays the greatest total fluorescence approximately 2.5 times greater than that with 0μl IPTG. All the cells had a greater fluorescence than that with 0μl indicating that it is essential for efficient GFP translation but at 20μl and 100μl total fluorescence is diminished.

This initially implies that the fluorescence is in fact reduced in the cells with more IPTG added, therefore we suspected there may be a toxic effect on the cells hence the OD was investigated.

Graph showing OD over fluorescence and demonstrates the suspected result; that IPTG reduced the growth of the BL21 cells during measurement over 4μl. At 20μl and 100μl the OD is reduced by approximately 50%, therefore must be taken into account when comparing efficacy of GFP expression.

This shows, most accurately, the comparison of the fluorescence of the BL21 cells with a spectrum of IPTG amounts added from 0μl to 100μl. This shows conclusively that with greater amounts of IPTG the fluorescence is increased. With 20μl and 100μl the fluorescence shows a steeper trajectory than at 4μl and with 0μl is significantly lowered. There is very little increase with 100μl from that seen with 100μl indicating that perhaps adding greater than 20μl is a waste of resources and has very little, or no, beneficial effect.

As the plotted graphs for each IPTG concentration do not show a plateau at any of the time points investigated we compared the gradient of the graphs to indicate the relative potential for fluorescence under each condition.

This bar chart compares the gradient of the lines plotted in the graph above in order to examine the relative potential of the transformed cells under the varied conditions. Only values obtained between 0.3 and 0.5 OD units were included in the creation of this chart as this is the OD between which ''E. coli'' demonstrates its exponential growth phase and would be expressing the transformed plasmid. This shows that the fluorescence increases exponentially from just over 1000 arbitrary units in cells with 0μl IPTG added to over 6 times as much relative fluorescence in those cells with 20μ added. However, this bar chart also demonstrates the same plateau effect, as seen in the line graph above, and supports the hypothesis that above 20μl more IPTG has no beneficial effect and is a waste of resources.