Latest revision as of 19:29, 17 October 2014

Imperial iGEM 2014

Safety

We submitted our iGEM safety form by September 1st and this was approved.

In the Lab

All our team members received safety training before being permitted to start working in the lab. This included training in fire procedures, waste disposal, accident reporting, cleanliness and good laboratory practice. This was all done in accordance with our Department of Bioengineering’s safety guidelines which can be found here.

All work carried out was within the biosafety guidelines established by Imperial College Safety Department: Site Genetic Modification Safety Committee which can be found here. UK regulations were adhered to.

The appropriate personal protective equipment was used which included lab coats gloves and where necessary safety spectacles.

Public and Environment

The Gluconacetobacter and E. coli strains used are not thought to pose any risk to the safety and health of the general public as they would not be able to compete outside the lab.

Furthermore the potential to misuse components of our project is limited. It may make being able to produce functionalised cellulose more accessible to the general public or 'DIY Bio' labs, perhaps persons with malicious intentions could functionalise with toxic proteins or other harmful substances. This, however is a problem shared by all iGEM and synthetic biology projects and not unique to our work.

Containment

As our project seeks to produce a useable biomaterial from our bacteria we paid special attention to issues of containment. The end product (the physical bacterial cellulose paper) will not contain any bacterial organisms.

Safety of the cellulose – bacterial retention after processing

Figure 1: The effect of the prolonged treatment with heat on the presence of the *G. xylinus* in the cellulose pellicle. Colonies recovered from: A) Wet untreated cellulose B) Wet untreated cellulose co-cultured with E. coli C) Cellulose treated with 60°C for 30 minutes D) Cellulose treated with 80°C for 30 minutes E) Cellulose treated with 60oC for 2 hours F) Cellulose treated with 80°C for 2 hours.

Figure 2: The graph shows the colony forming units of *G. xylinus* and *E. coli* recovered from the cellulose pellicle subjected to different processing methods. The cellulose was heated while submerged in H2O to prevent desiccation.

Figure 3: The graph shows the colony forming units of *G. xylinus* and *E. coli* recovered from the cellulose pellicle subjected to different processing methods. The cellulose was heated while submerged in H2O to prevent desiccation.

In order to analyse the safety of the bacterial cellulose, and its retention of G. xylinus, we have tested a number of processing conditions, of the wet unprocessed cellulose and plated it afterwards to obtain an idea if at all and how much of the bacteria remain in the cellulose pellicle after processing. We have also attempted a co-culture with RFP expressing E. coli cells, to observe how the E. coli embeds itself into the cellulose pellicle and if it is possible to extract it after processing.

The common processing method of cellulose in chemical engineering still remains treatment with NaOH, however we have also been testing the processing of the cellulose with heat alone, in order to be able to functionalise the cellulose fibres after processing.

The method of processing during the safety experiment is noted for each of the results. It is also important to note that most of the E. coli cells recovered were not expressing RFP (due to lack of selection pressure), so the E. coli counted for the results were identified by the colony morphology.

The ‘wet’ pellicle means the original cellulose produced by the G. xylinus. During all of the processing treatments the ‘wet’ pellicle was kept in water in order to avoid desiccation, to determine the effect of the heat alone. The ‘dry’ pellicle refers to the original cellulose produced by the G. xylinus that have been processed by the heat and drying.

Results

As expected the NaOH treatment killed all of the cells present in the cellulose and none of CFUs were recovered. Zero G. xylinus colonies were recovered from cellulose after virkon treatment, but one E. coli colony was recovered. This can be due the short treatment with virkon (10 min). More than anything this result highlights the importance of time when sterilising with virkon.

The heat treatment of the cellulose in its ‘wet’ and ‘dried’ state was able to kill all of the G. xylinus in cellulose after 60°C and 80°C degree treatment that was timed for 2 hours, since zero CFUs were recovered. It can also be observed that the same treatment with the 30 minute duration did not have the same effect and a number of CFUs of G. xylinus were recovered from both 60°C and 80°C 30 minute treatment.

The co-cultured cellulose with E. coli was subjected to processing treatments. From figure 2 it can be seen that in all of the heat treatments of the ‘wet’ cellulose pellicle that was kept in water, at least one colony of E. coli was always recovered, no matter the temperature or the length of the treatment. In contrast to this the data from figure 3 describe the ‘dry’ cellulose pellicle that was in addition to the heat treatment also dried at the same time. The treatment with 60°C for 30 minutes of E. coli cellulose co-culture showed recovery of one colony. It is possible that during this treatment the cellulose pellicle did not desiccate completely. All the other heat treatments, 60°C for 2 hours, 80°C for 30 minutes and 80°C for 2 hours, showed that no E. coli colony was recovered. This suggests the importance of desiccation of the cellulose during co-culture.

We can conclude that the G. xylinus and E. coli, can be removed from the cellulose pellicle by heating and drying alone. It is important to note that the removal of G. xylinus was dependent on the time of the heat treatment, with the 2 hours observed in this experiment showing as sufficient, with both 60°C and 80°C temperatures. The E. coli removal from the cellulose pellicle did not seem to be dependent on the temperature; instead it was based on the dryness of the cellulose. Dried cellulose is not able to support E. coli and the bacteria cannot be recovered.

We suggest for successful removal of G. xylinus and E. coli from the cellulose, a drying and heating treatment with 80°C for 2 hours.

Team:Imperial/Safety

From 2014.igem.org