Team:Brown/Parts/In Progress

1 BBa_K124003 – This part is the DNA received from John Mekalanos’ Lab at Harvard Medical School, pvj4. The sequence, 1273 base pairs in length, codes for three different proteins. These three proteins, commonly known as the S, R, and Rz genes, encode for cell lysis. The three genes packaged together are regularly called the “Cell Lysis Cassette.” The S gene is a holin and the R gene is an endolysin. Little is known about the function of the Rz gene in cell lysis but it is involved in many cell lysis processes.

• Biobrick Primers containing proper Prefix and Suffix:
• Prefix: 5’ atg aaa tca atg gac aaa atc tca act ggc ATG AAA TCA ATG GAC AAA ATC TCA ACT GGC 3' Melting Point = 58 deg C
• Suffix: 5’ GTT TCT TCC TGC AGC GGC CGC TAC TAG TA cta tct gca ctg ctc att aat ata ctt c 3' Melting Point = 56 deg C

2 BBa_k124014 – Received from Ry Young at Texas A&M. S105 is a mutant of the single S gene in the Cell Lysis Cassette. It’s purpose is to create a hole in the cellular membrane, hence the name “Holin.” This variation of the S gene induces lysis at a faster rate than the Wild Type. The length of time required for complete lysis is much shorter due to the deletion of the antiholin product, S107. The first 6 base pairs of the Wild Type sequence are the N-terminus residues of S107. S105 does not have these first 6 base pairs enabling it to be more efficient at membrane-permeabilizing activity.

• Biobrick Primers containing proper Prefix and Suffix:
• Prefix: 5' GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ATGCCAGAAAAACATGACCTGTTGGC 3' Melting Point = 58 deg C
• Suffix: 5' GTT TCT TCC TGC AGC GGC CGC TAC TAG TA TTA TTG ATT TCT ACC ATC TTC TAC TCC GGC 3' Melting Point =59 deg C

3 S105 Cassette - This part contains the S105 Holin gene as well as the R and Rz genes that complete the lysis cassette. The R and Rz genes are the same sequences as the R and Rz on pVJ4 (part BBa_k124003) received from the John Mekalanos' Lab. We used the Prefix primer of S105 and the Suffix Primer of PVJ4 to Biobrick the part.

• Biobrick Primers containing proper Prefix and Suffix:
• Prefix: 5' GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ATGCCAGAAAAACATGACCTGTTGGC 3'Melting Point = 58
• Suffix: 5’ GTT TCT TCC TGC AGC GGC CGC TAC TAG TA cta tct gca ctg ctc att aat ata ctt c 3' Melting Point = 56

4 The R Gene - The second gene of the Cell Lysis Cassette. It is an endolysin causing lysis of the bacterial cell. The gene is 477 base pairs in length.

5 The Rz Gene The third gene in the Cell Lysis Cassette. It is 462 base pairs, however, little is known of its function in cell lysis. It is included in many cell lysis methods but has yet to be characterized sufficiently.

Composite Parts

Mercury Sensor BBa_I728456 (Mercury Inducible Promoter) coupled with BBa_K124003 (PVJ4 Cell Lysis Cassette). The current Arabinose Inducible Promoter is replaced with a Mercury Promoter. In the presence of Mercury in a water sample, the three lysis genes, S, R, and Rz genes will be expressed.

Xylene Sensor BBa_I723020 (Pu) and BBa_K124003 (PVJ4). Transcription activated in the presence of environmental xylene.

Arsenic Sensor BBa_J33201 (E. coli chromosomal ars promoter with arsR repressor gene) and BBa_K124003 (PVJ4). A method for detection of Arsenic in water supplies.

LacI BBa_R0010 and BBa_K124003 (PVJ4). When lactose or IPTG is present, lacI is unable to bind and prevent transcription.

T7 BBa_I719005 and BBa_K124003. Part BBa_I719005 is the T7 promoter. Proteins are expressed at high levels in the presence of T7 Polymerase.

The AND Gate - The individual S and R genes can be placed under different promoters, creating an AND Gate. For example, the S gene can be placed under an Arabinose promoter while the R gene is placed under an IPTG (isopropyl-beta-D-thiogalactopyranoside) promoter. (The IPTG promoter induces gene expression of genes under the control of the lac operon.) The S and R genes will not be expressed unless both "toxins" are present. If one of the toxins is present and the other is not, there will be little to no gene expression of the Lysis genes.