Difference between revisions of "Part:BBa K3409010"

Latest revision as of 20:53, 25 October 2020

Microcin PDI cluster genes for McpM, McpI, McpA

Contains the elements necessary to express the three first genes from the Microcin PDI gene cluster (mcpM: coding for microcin PDI, mcpI: coding for the immunity protein, mcpA: coding for the activator protein). All three genes are under the control of the same promoter and the sequence is organized as follows: a promoter (BBa_J23111), an RBS (BBa_J61127), the CDS for McpM-His (BBa_K3409003), an RBS (BBa_J61130), the CDS for McpI (BBa_K3409004), an RBS (BBa_J61118), the CDS for McpA (BBa_K3409005) and a double forward terminator (BBa_B0015). Note that it must be expressed with the two other genes from the cluster (mcpD and mcpB) for secretion of McpM. Will constitutively generate McpM, McpI and McpA.

Usage

This part is used to produce Microcin PDI, its immunity protein for the producing strain as well as the activator protein. For microcin PDI to be effective against a target strain it must be excreted.

Biology

Attention must be given to the quantity of McpM and McpI produced as there must be a balance between both. Without the immunity protein, the producer strain will die.

Characterization

NOTE: In this section: BB1 = part BBa_K3409010 - BB2 = part BBa_K3409011 – BB3 = part BBa_K3409012

PCR AMPLIFICATION OF PARTS

Once received, the synthetized fragments are resuspended and amplified via polymerase chain reaction (PCR) to obtain a higher quantity of DNA. Every PCR sample is verified by gel electrophoresis to analyze the PCR results. First, the appropriate bands for BioBrick 1 (1.5 kb) , BioBrick 2 (1.4 kb) and BioBrick 3 (2.4 kb) amplified with the primers for Hifi Assembly method, are present at their expected size (Figure 1A). Then, the appropriate bands for BioBrick 1 (1.5 kb), BioBrick 2 (1.4 kb) and BioBrick 3 (2.4 kb) amplified with the primers for BioBrick Assembly method, are present at their expected size on Figure 1B.

INSERTION OF BBa_K3409010 IN PLASMID AND BACTERIAL TRANSFORMATION

Concerning the test the insertion of BioBrick 1 into the pSB1A3, plates have been incubated at 30°C and 37°C degrees. Both plates with the sample (Figure 2 – on the right), which contained only BioBrick 1, do not show any colony. However, on the control plates (Figure 2 – on the left) few colonies can be counted. Which suggest that bacteria could not survive due to the expression of McpM of BioBrick 1. Thus, McpM seems to toxic for the bacteria.

MCPM PRODUCTION VALIDATION

Extraction, purification and validation of McpM production

To validate the production of McpM, the protein needs to be extracted and purified from the cellular culture via Ni2+ chromatography column and further revelation via SDS-Page and staining technique.

Expected results

The McpM size is 8 kDa. Therefore, we expect that the in the lane of the elutions a band appear at the size of 8 kDA. In the first lane, this band should be dark and be less intense in the lane where Elution 2 and 3 have been loaded. We also expect to have few bands in the flow through corresponding to potential waste material. Regarding the lysate, the same bands as the flow through can appear, plus one at 8 kDa. Finally, in the lane of the Laemmli no bands should appear

Sequence and Features