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RESULTS

F. solani Growth

The fungus in liquid culture is incubated at 27°C and 120 rpm for 7 days.

Optimisation of F. solani Transformation

The plasmid pCAMBIA 1301 contains the GUS reporter system and the antibiotic resistance genes for hygromycin and kanamycin. For transformation of this into F. solani, the protocol adopted was electroporation.

Fungal culture along with DNA and a negative control with no DNA were subjected to electroporation. They were subsequently streaked onto hygromycin B slants. After a few days there was no growth in the negative control as expected and the other slant showed growth indicating successful transformation.

Figure 1: Slants with the transformed cultures

Cloning of BioBricks

The first step in the cloning of our BioBricks into the plasmid pCAMBIA 1301 was the isolation of the plasmid from a stock E. coli TOP10 culture. This glycerol stock was plated and then inoculated in LB medium from which plasmids were extracted through a kit-free midiprep. The isolation of plasmids was confirmed through agarose gel electrophoresis. The Invitrogen^TM 1 Kb Plus DNA Ladder was used in all electrophoresis experiments.

Figure 2: pCAMBIA 1301 Isolated through Midiprep

The isolated plasmids were then subjected to a double-digestion using the restriction enzymes PstI and AflII so the eGFP construct could be cloned into it. Restriction digestion was seen to have worked and the band of the right size (8,968 bp) was excised from the gel and eluted.

Figure 3: Double-Digestion of pCAMBIA 1301

The two inserts containing eGFP and the enzyme strictosidine synthase (STR) respectively were PCR-amplified from the product obtained from IDT. The PCR products were run on a gel to confirm amplification. Having confirmed that amplification had worked, both products were subjected to double-digestion. The construct containing STR was digested with EcoRI and PstI while the construct containing eGFP was digested with PstI and AflII. All restriction enzymes were obtained from New England Biolabs.

Figure 4: PCR-Amplification of the Inserts

An overnight ligation reaction using T4 DNA Ligase was set up in which the eGFP insert was cloned into the plasmid backbone. The reaction mixture was then transformed into ultracompetent E. coli TOP10 cells which were then plated on LB Media containing kanamycin (50 ug/ml) and incubated overnight. This yielded over 25 colonies. A plasmid self-ligation was set up as a negative control. This reaction mixture, when transformed into ultracometent TOP10 cells, yielded no colonies that survived on media containing kanamycin.

Liquid cultures of 10 tranformant colonies were set up and plasmids were isolated from them through a miniprep protocol. The presence of plasmids was confirmed through agarose gel electrophoresis.

Figure 5: Plasmids Isolated from 10 Transformant Colonies

To confirm that the isolated plasmids contained the eGFP insert, double-digestions were set up in which the plasmids isolated from each colony were digested using PstI and AflII so as to release the insert. The resulting reaction mixture was run on a gel to confirm the presense of the insert.

Figure 6: Insert Release to Confirm Cloning

For further confirmation, PCR reactions were set up using plasmids isolated from each colony as the template and forward and reverse primers specific to the insert. Bright bands were observed for all colonies.

Figure 6: PCR to Confirm Cloning

Part Characterisation

INTRODUCTION:

The Cauliflower Mosaic Virus (CaMV) promoter 35S is a strong constitutive plant promoter (Odell et al. 1985; Benfey and Chua 1989). Studies indicate that this promoter can be used to express genes in E. coli.( Assaad and Signer 1990). This can be advantageous as expression can be verified rapidly and with considerable saving of time and manipulation as this promoter is typically used with plant constructs.

We have validated the expression of an enhanced CaMV 35S promoter fused to a hph gene cassette in E. coli. This is a part of the plant vector pCAMBIA 1301 which has been used for our experiments. Details of the usage of this plasmid can be found here.

MATERIALS AND METHODS:

TOP 10 with the pCAMBIA 1301 plasmid was grown on LB agar. 100 ul of an overnight culture (5 mL) developed from a single colony was used to plate on 10 plates with varying concentrations of hygromycin B (100-1000 ug/ml).

RESULTS:

E. coli had no visible growth on 50 ug/ml of hygromycin B and this was found to be the minimum inhibitory concentration. This value is consistent with other studies. Our transformed cells containing the hph gene conferring resistance to hygromycin B under the control of the CaMV 35s promoter were resistant to up to 200 ug/ml of hygromycin B.

Figure 7: TOP 10 + pCAMBIA 1301 in 100ug/ml of Hygromycin

Figure 8: TOP 10 + pCAMBIA 1301 in 150ug/ml of Hygromycin

Figure 9: TOP 10 + pCAMBIA 1301 in 200ug/ml of Hygromycin

Figure 10: TOP 10 + pCAMBIA 1301 in 300-1000ug/ml of Hygromycin

CALCULATIONS:

We calculated the initial number of cells to be 7.2 x 10⁸ cells/ml based on the OD.

At 50ug/ml of hygromycin B, the number of cells =7.2 x 10⁸ cells/ml

At 100ug/ml of hygromycin B, the number of cells=7.2 x 10⁸ cells/ml

At 200ug/ml of hygromycin B, the number of cells=(say 1/5th of what we had earlier) 9 x 10⁷ cells/ml

Figure 11: %Sensitivity of cells in response to varying hygromycin B concentration

Part Validation

We created a part ( BBa_K3163002) such that eGFP is driven by a CaMV 35s Promoter and flanked by NOS terminator which is already present in pCAMBIA 1301. We cloned this part into pCAMBIA which replicates in Fusarium solani and E. coli. Here, we have transformed the plasmid in to E. coli TOP 10 and performed fluorometry studies to confirm the presence of GFP. analysis. Higher fluorescence in the culture containing our part confirmed the presence of GFP and this was found to be statistically significant by ANOVA analysis. Characterisation of BBa_K3163002 in E. coli is reported in the figure given below.

Figure 12: Normalized Fluorescence with respect to Empty Vector (pCAMBIA 1301) in E. coli.