BGs drew increasing interest in the recent years particularly aiming to be used as an immunostimulant or as a drug delivery carrier. For that, a method based on E lysis gene has been established. The E lysis gene is coded by a temperature sensitive promoter, so at the correct time after reaching the critical biomass, the temperature of the viable order GS1101 is adjusted and for that, the internal component of the cells are lysed and the cells turn to be unviable or BGs. However, using such genetic based tools might still have some type of risk. Different genetic elements could by one or other routes be passed to our genetic material and might be harmful. For that, it is important to find alternative methods for BGs preparation not based on the use of the genetic elements. Amara et al. (2013), and for the first time introduce a fully described and optimized protocol for BGs preparation. The protocol is based on using different tactics for reaching the target of the BGs. The cells have been aged to give a thicker cell wall. The chemicals have been used in two concentrations showing minimum effect on the bacterial cells. They are the MIC and the MGC. Moreover, Experimental Design tools which are represented in Plackett–Burman have been used to map the best conditions and guarantee the best production for the BGs. The BGQ is evaluated using light and electron microscope as in Fig. 2. In this study, precisely, we follow the original protocol for preparing the BGs from another E. coli strain the E. coli JM109. The BGs preparation that has been summarized in a protocol enable better propagation upon following or those conducted by any. E. coli JM109 prove to be more sensitive to SDS than E. coli BL21 (DE3), where the (+1, −1) values were 0.24mg/mL and 0.03mg/mL of SDS respectively. The MIC and MGC of E. coli JM109 for each of the NaOH and H2O2 have been as same as those of E. coli BL21 (DE3) and are represented by 0.0138N and 0.00231N (+1, −1) for NaOH and 40.8μL/mL and 5.83μL/ml (+1, −1) from 30% H2O2 for H2O2 respectively. This might be an indication about the cell wall variation; a tool that might be used in future for the determination of the competent cells rigidity and transferability. In the case of CaCO3 the used amount of +1 value was 1.05μg/mL while −1 value was 0.35μg/mL. The twelve experiments which contain either the +1 or −1 value for each variable in each experiment in random arrangement have been conducted at the same time to get the best results and to enable the best possible comparison. The BGQ has been given for the 100% quality as 10, while ten cells have been evaluated as either bad or good. This will decrease the range of the differences if we use %. Unexpectedly, E. coli, which is more sensitive to the SDS than E. coli BL21 (DE3) gives better results with most of the experiments. Nine experiments give the number 10 out of the twelve experiments. Two give the number eight and only one gives the number which means very poor preparation. In experiment twelve which give the number SDS and CaCO3 have been used in the +1 value. It is clear that SDS and CaCO3 might coordinate to damage the cell wall. Logical analysis of the differences was done and analysed why experiment twelve gives quality BGs? To understand what happened to make the cells in experiment twelve completely damaged a special comparison between the experiment twelve and experiment one and three has been generated and extracted from the main Plackett–Burman Table 1 and summarized in Table 5. In experiment twelve, SDS and CaCO3 are the only factors that might affect the cells severely in quality while they have been used in their +1 value. In experiment number three SDS, CaCO3 and NaOH have been used in their +1 which might be responsible for the loss in the quality. In experiment number one NaOH has been used in −1 which might be responsible for obtaining the highest quality score. If similar variables in experiment one and three compared with experiment twelve are ignored, one variable (+1 H2O2) in experiment one and two variables in experiment three (+1 NaOH and −1 Shacking rate–Temperature) are still different. It must be that, low temperature and shaking rate in the presence of +1 SDA has a negative effect on the bacterial ghost preparation in the condition of experiment twelve. H2O2 (−1) if used in the condition of experiment number twelve will also reduce the BGs quality. Additionally, low temperature and shaking rate might enhance SDS (if represented in high amount +1) to damage the cells. For doing more unbiased analysis, the Main effect of the variables have been determined as in Fig. 1. The main effect clearly supports our argument in the logical analysis of the data. Clearly, H2O2, NaOH and Shaking rate–temperature positively affect the cells quality when used in higher concentrations SDS proved in the experiment number twelve that it is able to negatively affect the BGs quality. CaCO3 does the same but in a stronger way as in the above Figure. In the previous study made by Amara et al. (2013) who used the same tools each of the NaOH and Shaking rate–Temperature negatively affects the BGs quality. Here and unexpectedly, they are positively affecting the BGs quality. That might explain the fact that both E. coli strains are different in their behaviors and responses to various treatments and that E. coli JM109 is more sensitive to the changes in the chemical compounds as proved by the MIC and MGC in a positive way. It seems that JM109 might have more ability to neutralize the effect of the SDS if a larger shaking rate has been used. This might enable faster release of the internal protein content, which will react with the SDS and neutralize it. But in the case of low temperature and shaking rate it might be that this condition makes the cells unable to get rid of their protein content and the level of the SDS remains high and continues in its attack on the cell wall. Low amount of protein reading using a spectrophotometer might be apparently measured in the case of higher temperatures and shaking rate (even expected high release of the protein) might be due to that the SDS-Protein complex is not detectable at 280nm or precipitated. An observation must be investigated in future studies. Meanwhile, one should compare our data in this study with those in Amara et al. (2013), for a clearer image about the effect of various used chemical compounds on the different E. coli strains used. Main effect analysis, is a simple but a powerful tool for determining which variables positively and which negatively affected the BGs quality.