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Applications of BBa_M36459

To accomplish the goal of induced cell lysis, various cell lysis DNA sequences were examined on the BioBricks part registry. One particularly intriguing DNA sequence included three encoded proteins, an S105 protein (Holin), an R protein (Endolysin), and an Rz protein. These three proteins combine to cause total cell lysis. This sequence was edited to avoid palindromes and repeating sequences greater than or equal to 10 base pairs. Holins consist of an extremely diverse group of proteins that all lead to cell lysis. Holins are unique in that they are “time-specific”; they accumulate over time and then lyse the cell membrane all at once. The endolysin kills the cell by disruption of the cell wall. This is important to include in our sequence since E. Coli has a cell wall. The Rz protein helps the holin perforate the cell membrane to cause lysis. All three of these proteins are necessary for complete cell lysis of E. Coli.

A Bicistronic Design (BCD) was used for the first Ribosome Binding Site (RBS) in order to accurately predict transcription levels. This is especially important when dealing with cell induced death as an unexpected high level of transcription could lead to premature cell death before enough E. Coli was produced to do the testing. Thus, a BCD with a relatively low strength and extremely low variance was chosen for the DNA construct. The Endolysin and Rz protein were both paired with a Monocistronic Design (MCD) as there is too much similarity in the sequences of the BCDs to synthesize multiple in the same construct. However, the MCDs chosen had low expression and low variance as well. The MCDs and BCD were found using the supplementary data table to the paper “Precise and reliable gene expression via standard transcription and translation initiation elements”. For the BCD associated with the holin protein, we chose pFAB1692, with a relative strength of ~75 and standard deviation of ~3. For the MCD prior to the Endolysin, we chose apFAB706, with a relative strength of ~66 and a sd of ~2. And finally, for the MCD placed before the Rz protein, we chose apFAB720, with a relative strength of ~66 and a sd of ~6.

The terminator used for the cell lysis DNA construct was classified and rated in the journal article "Measurement and Modeling of Intrinsic Transcription Terminators”. It was the second strongest terminator that was tested with ~99% termination efficiency. Since this terminator is being placed at the end of our 3 protein sequence, there is no reason to use a weak terminator.

The cell lysis actuator was paired with a pre-proven sensor that is induced by Rhamnose and suppressed by Glucose. This sensor sends a signal to the actuator using Polymerase per Second (PoPS). The layout of the actuator will be First RBS, First GOI (Holin), Second RBS, Second GOI (Endolysin), Third RBS, Third GOI (Rz Protein), Terminator. This is relatively basic setup and each part comes directly after the previous part; there is no untranslated region within our sequence.

Our plasmid was placed in Escherichia coli as it is the easiest to test the cell lysis actuator in and it has the most practical application for the overall research idea of having dangerous bacteria lyse upon exposure to the environment. The plasmid is Kanamycin resistant since making the plasmid Ampicillin resistant could interfere with the lysis actuator. Ampicillin works by creating pores in the cell membrane leading to cell lysis, similarly to how the 3 protein sequence in the lysis actuator causes cell death. Kanamycin causes cell death by interfering with the 30S subunit in prokaryotic ribosomes. It is unknown whether Ampicillin would actually interfere with the cell lysis actuator designed.

Process:

Upon receiving the lysis actuator contained in a pJ821 plasmid from DNA 2.0, the plasmid was transformed using Calcium Competent cells prepared a month prior. Transformation was done twice by Whitmeyer and twice by Lewis to create a sham control. Unfortunately, none of the transformed colonies grew as expected as 2 did not grow at all and 2 were infected, determined to be fungal based on color and growth patterns. This was determined to be a problem with the Calcium competent cells. The procedure was repeated but this time with Top 10 cells to nearly eliminate the chance of failure due to the cells. Also, to control whether the cell lysis actuator was the problem (eg. it kills the cells before they are able to form colonies), an identical transformation with p81 plasmid was performed. P81 is also kanamycin resistant and compatible with E. Coli. The transformation was successful with both the pJ821 and p81 plasmids as both colonies grew successfully on LB + kanamycin plates in relatively equal numbers. Three colonies from both the pJ821 and the p81 plasmid were picked and allowed to grow up in liquid culture containing LB + kanamycin. After the cultures had grown up they were made into glycerol stocks and the remaining culture was used to perform test number 1.

There were four tests performed with our cell lysis actuator and they are outlined below. Results from each of these tests may be viewed in the results section of the paper. The initial test performed using three different colonies from both the experimental cell lysis actuator (pJ821) and a control, p81, to test whether or not the cell lysis actuator worked. The results would be confirmed by the response from p81, a plasmid that contains the same proven sensor as pJ821, but also a proven GFP actuator. 3 ml of leftover liquid culture from each of the colonies grown up were used (3 containing the experimental pJ821 cell lysis plasmid, 3 containing the p81 control plasmid). Each group of 4ml media was divided into 2 groups of 1.5 ml each. One of the 1.5 ml groups from each colony was given 15 uL rhamnose to create a 1000 uM solution of rhamnose while the other 1.5 group was given nothing. In the end, there were three 1.5 ml tubes containing the pJ821 + Rhamnose, 3 containing just pJ821, 3 containing p81 + Rhamnose, and 3 containing just p81. This test was done for qualitative reasons only as there was not enough media to create a strong test. However, this test would be able to allow us to see if our actuator was actually working.

The second test performed was a logarithmic scale of different amounts of rhamnose to check what dose would be lethal with the cell lysis actuator. This would be done using concentrations of rhamnose of 0 uM, 5 uM, 10 uM, 100 uM, 1000 uM. A liquid culture was grown up using glycerol stock 1 of the cell lysis actuator in 100 ml of LB + Kan. The culture was allowed to grow overnight in a 37 degree shaker incubator with the shake speed set to 275 rpm. The media was then distributed into 25 falcon tubes containing 2 mL of media each. The desired rhamnose was then added to each of the 25 falcon tubes (5 tubes for each of the 5 concentrations of rhamnose mentioned earlier in the paragraph). These tubes were then placed in a 25 degree shaker incubator with the shake speed placed on 200 rpm to allow the mixture to remain well mixed without significant E. Coli growth. The cell density of each culture was read the next day using a spectrophotometer.

The third test performed was done to provide a more accurate understanding of exactly what dose would be lethal when paired with our cell lysis actuator. Knowing that 5ml had a significant impact on the cell density with a non-significant decrease with a greater amount, it was decided to hone in on the 0 to 5 uM rhamnose area. This was done with the same process as the second test, with 5 tests for each value of rhamnose, 0 uM, 1 uM, 2 uM, 3 uM, 4 uM, 5 uM. The liquid culture was prepared and the rhamnose + actuator were stored in identical conditions. The only difference was that the values were read using a 96 well plate reader rather than a spectrophotometer.

Knowing the lethal doses of rhamnose was a critical first step for understanding the cell lysis actuator. However, it is also important to know the time period the actuator takes to work. This test was performed using 36 samples – 12 grown from a recently picked colony, 12 grown from glycerol stock 1, and 12 grown from glycerol stock 2. This was done to make sure the glycerol stocks were not affected in any way compared to the colonial cultures. Within each group of 12, 3 were given no rhamnose, 3 were given 1 uM rhamnose, 3 were given 10 uM rhamnose, and 3 were given 100 uM rhamnose. The amounts of rhamnose were varied so the action of different amounts could be compared over time. The 36 cultures with rhamnose added were placed in the 96 well plate reader along with a blank. The optical densities of the cultures were then measured every half hour for 12 hours.

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