Team:Peking/Project/Application/PreservationOptimization

From 2010.igem.org

Introduction

Biosensor and bioreporter as indicator of chemistry pollution has been widely applied in many fields. But traditional bioreporters are not practical when it comes to in field application. One crucial factor that restricts the in field application of bioreporter is the preservation condition of the bacteria. It is quite impractical to cultivate certain strain of genetically engineered bacteria to achieve harvest in the field. So the best solution is the optimization of preservation condition, which can be applied in the field easily and quickly. We are trying to fulfill two goals: one is to preserve the bacteria in a powder for or a glassy form at non-costly condition, the other is to make the bacteria able to be recovered as quickly as possible and maintain a relatively high activity. By this way, bacteria reporters would be quite available in the field.

There are some alternatives to choose from, among which are the germination of spores to vegetative cells, low temperature preservation (-20°C), and freeze dried method. Unfortunately, all of them are not available, due to the low survival rate, or the long time to recover. It is now supposed that the most effective way is to ambient dry with a preservation media. Previous work indicated that a kind of sugar, trehalose, stabilizes membranes and proteins in the dry state, most likely by hydrogen bonding to polar residues in the dry macromolecular assemblages. This direct interaction results in maintenance of dry proteins and membranes in a physical state similar to that seen in the presence of excess water. Trehalose has an advantage over other kinds of sugars for it has a relatively high glass transition, so the bacteria can maintain a glassy form when temperature and humidity varies. It is also important to produce in cell trehalose by the bacteria spontaneously, which involves certain gene expression and an osmotic press induction. In E. coli, the otsA and otsB genes are responsible for trehalose biosynthesis from UDP glucose. These genes encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. And high concentration of NaCl solution induces the expression of intracellular trehalose.

So the protection media should contain trehalose at least. And previous works also indicated that high concentration of polyvinylpyrrolidone (PVP) contribute to a relatively long time of maintenance. Previous work also suggested that adding an ounce of two kind of salt, sodium ascorbate and sodium glutamate, may have an effect on the preservation result. Our main task is to see whether these two salts are able to improve the effect of preservation, and to determine if a best result can be achieved by a higher concentration of trehalose!

Method

• We prepared 4 types of protection media (Table-1).

In sum, we constructed two strains of E.coli bacteria, BL21 bearing standard plasmid with T7 promoter + BBa_E0840 as an insert, which will express GFP when induced by IPTG; the other strain was developed from DH5 alpha, bearing plasmid with Constitutive Promoter+MerR gene and PmerT Promoter+BBa_E0840, which will express GFP when induced by Hg (II).

We firstly cultivated single colonies of each strain for 8 hours at 37°C. Then resuspend in 5 ml LB medium containing 2% NaCl for another 8 hours (for preconditioning under osmotic stress, and the bacteria in the NaCl media grows too slow). The cells were harvested by centrifugation (6 min at 3,800×g). Preliminary tests had indicated better survival resistance of stationary phase cells compared to cells from the exponential growth phase. Bioreporter preservation by vacuum drying stationary phase bioreporter bacteria (salt-preconditioned or not) were resuspended in different drying protection media to achieve culture optical density at 10. Here, we use four kinds of protection media. Ten microliters of the cell suspensions in protection media was applied to an Eppendorf tube and subsequently vacuum-dried at ambient temperature using water pump for about 10h. The resulted samples were glassy like rather than powder like, stored at 4°C.

• We applied two methods to evaluate how the samples recover.

1. Colony-forming units and determination of total cell counts for determination of colony-forming units (CFU). We added 1ml of ddH2O on the sample to recover for about 30min in 37°C, and then added 100ul of the media to 900ul ddH2O, followed by applying 100ul of the dilution plated on LB agar plates. The plates were incubated overnight at 37°C before counting the number of colonies.

2. We applied 1ml of LB media with inducer (1uM IPTG or 10^-7 uM Hg) to each sample for recovery, cultivating for about 10h. Then the cells were harvested by centrifugation (13000* 5min), and resuspended with 500ul PBS. Then GFP intensity was recorded by Microplate Reader, every hour to monitor the recovery situation. We also test the GFP intensity under different density of inducers to draw a dose response curve. We supplied LB media with different concentration of inducers (for IPTG from 10^-3 to 10^-7 and for Hg from 10^-5 to 10^-9), and cultivate for about 15 to 25 hours. For comparison, the same procedure was performed every time with fresh cells from the stationary phase of an overnight culture. Cell activities were characterized by the time until the color development started (referred to as the response time (RT)), the maximum corrected A600 achieved (the response intensity, RI).

Results

Short term preservation (20 days):

To identify the best preservation conditions for the bioreporter activity maintaining, we used uM Hg (II) or uM IPTG to induce the cells that had been preserved in different protocols. Fresh cells harvested in the stationary phase on each sampling day served as control.

After 20 days storage at 4°C, the samples were then recovered by incubating for 20h in LB media at 37°C. Figure 1-1 shows the OD values of the samples. The overall growth situation of samples in all different protection media was similar, while the T7p-GFP strains were more vigorous. The difference between the two trains more likely resulted from the inherent differences of the cells, rather than the effects of different protection media. So it can be concluded that the four different media have similar effects on the cells considering the growth situation. Figure 1-1 shows that the OD values of cells with or without inducers are approximately the same. So the inducers had little to do with the growth conditions, in other words, such concentration of inducer neither represses nor facilitates the growth of the cells.

Figure 1-1 The samples were incubated in LB with/without inducer for about 10 hours, and then measured the OD value of each sample to compare the difference of growth situation with or without inducer

Figure 1-2 shows the response intensity of the samples, the control group was samples without inducer supplied. The T7p-GFP strain with PT68(-) media failed to express GFP. For the rest of the samples, the T7p-GFP strain had a much higher expression level of GFP than MerR-GFP strain. For T7p-GFP strain, sample of PT34(-) had the highest intensity, followed by PT68(+) and PT34(+). For MerR-GFP strain, the difference of expression level between samples with inducer and the control groups is slight, so it is hard to compare. Depending on the expression level of T7p-GFP strain, PT34(+) protection media has the best effect, while PT68(-) has the worst effect for cell protection. From Figure 1-2, the highest intensity of every sample was also indentified (see Table 1).

Figure 1-3 shows the value of GFP/OD, representing similar overall trend with Figure 1-2.
Figure 1-2 The samples were incubated in LB with/without inducer for about 10 hours, and then measured the GFP intensity of each sample to compare the difference of growth situation with or without inducer

Figure 1-3 With OD value and GFP above, we could determine the GFP/OD value of each sample.

Figure 2-1 shows variations of all the samples OD value with incubation time. The OD values were measured after 7-10 hours. For MerR-GFP strain, the sample PT34(-) already began to grow after 7 hours, according to the OD values, approximately 2 in 7 hours and remaining the same for the next three hours , so their lag time esd actually shorter than 7hours. The lag time for PT34(+) and PT68(+) were 8 hours, while the lag time for PT68(-) was about 9 hours. For T7p-GFP strain, the sample PT34(-) remained a very low OD value through 7 to 10 hours, so it’s lag time would expect to be well longer than 10 hours. Other samples’ OD values gradually increased to about 0.05 to 0.2 or 0.25.

Figure 2-2 shows the variation of GFP intensity with incubation time. The T7p-GFP strain’s expression level remained very low through the whole process. Their response times were longer than 10 hours. For MerR-GFP strain, intensity of PT34 (-) and PT68 (+) were already very high in 7 hours, so their response were shorter than 7 hours. The response time of sample PT34 (+) was 8 hours and that of PT68 (-) time was 8 hours. Figure 2-3 shows the variation of GFP/OD value with time, and it reflected a similar results.