pP6 Intro: Finding Strong Promoters in E. coli

Goal of the Experiment

Your objective is to create and test a library of synthetic promoters to discover variants with high transcriptional strength. The goal is to create a family of distinct promoter parts that span a range of strengths, suitable for use in multi-gene constructs without sequence redundancy or recombination risk.

Getting Started in the Lab

To perform this experiment, you’ll need access to a fully equipped molecular biology lab. This includes basic tools like a set of pipetman, a thermocycler, an incubator, a shaker, and standard molecular biology reagents. You’ll also need the pJ12 plasmid template and two synthetic oligos ordered from IDT that introduce the promoter library variability.

Before going to the bench, download and fill out the pP6 LabSheet Workbook, which will guide your workflow:

pP6 LabSheet Workbook

To prepare:

1. Make a copy of the LabSheet (as a Google Sheet).
2. Enter your name and assigned ID on the ‘Entry’ worksheet.
3. Either print out the sheets from ‘PCR’ through ‘MiniSeq’ or bring them on a laptop, tablet, or phone.
4. Watch the demo video for each task before performing it in the lab.

These sheets contain the bench-level instructions you’ll follow throughout the experiment.

Background: Transcription in E. coli

Figure: Transcription in E. coli. RNA polymerase binds the promoter at the -35 and -10 sites, initiates at +1, and transcribes a coding sequence (CDS) into mRNA with an RBS. The transcript ends at a terminator sequence.

When RNA polymerase binds to a promoter in E. coli, it begins transcription at the +1 position, synthesizing an mRNA strand. This mRNA includes a ribosome binding site (RBS) and a coding sequence (CDS). The -10 and -35 sequences upstream of the +1 site strongly influence transcription strength.

The J23100 Promoter Family

Years ago, a standard set of synthetic promoters was developed by mutating a parent sequence called J23119. This set, known as the J23100 promoter family, provides a spectrum of expression strengths and has proven very useful for fine-tuning gene expression.

These promoters are ideal when you want to scan through different expression levels of the same gene using well-characterized sequences. Each member of the family was created by introducing mutations into the -35 and -10 boxes of J23119, while keeping the flanking sequence constant.

Figure: The J23100 promoter family. Variants were created by mutating the -35 and -10 regions while keeping flanking sequences constant. Fluorescence and sequence alignment show relative expression strengths.

Why Build New Promoter Libraries?

Although the J23100 family is widely used, it has some limitations:

• All promoters share a nearly identical sequence backbone, making them prone to homologous recombination when used together in a single plasmid.
• Repeating these sequences interferes with PCR-based edits and makes cloning less reliable.

To overcome these issues, we aim to build multiple promoter families with similar activity ranges—but based on entirely distinct sequences. These new libraries can be used together safely in the same construct and facilitate more advanced synthetic biology designs.

The pP6 Library Design

The pP6 experiment is designed to generate a sixth-generation promoter library following this strategy.

We retain only the minimal motifs required for strong σ⁷⁰ recognition and randomize all other surrounding bases. This allows us to create new promoters that are strong, functional, and non-redundant.

• Fix the -35 and -10 consensus motifs.
• Randomize all other positions.

Figure: σ⁷⁰ consensus promoter. The sigma factor of RNA polymerase recognizes the -35 (TTGACA) and -10 (TATAAT) boxes and initiates transcription at the +1 site. This image shows the promoter region unwound and engaged by polymerase.

We also add 4 random bases upstream and downstream, yielding 31 degenerate positions in total.

Construction File

We use a "construction file" to formally describe how DNA parts are assembled. Each line represents a biochemical reaction step using standardized syntax.

The pP6 promoter library is built using a two-step process involving PCR and Golden Gate assembly.

We will use a technique called EIPCR (Enzymatic Inverse PCR), which involves amplifying the entire plasmid by PCR. Each PCR primer includes a constant 3′ annealing region and a 5′ overhang containing degenerate bases (the N's). The 'N' effectively means "pick a random base from A, T, C, or G for this position". This introduces variability into the unconserved promoter regions:

Primers:

>P6libF2
CAGTAggtctcgATAATNNNNNNANNNNGTTAGTATTTCTCCTCGTCTAC

>P6libR2
CCAAAggtctcgTTATANNNNNNNNNNNNNNNNNTGTCAANNNNGAACCCAGGACTCCTCGAAGTC

Construction of pP6

# PCR reaction
# step      primer1     primer2     template     product
PCR         P6libF2     P6libR2     pJ12         P6

# Golden Gate assembly
# step          dna         enzyme     product
GoldenGate      P6          BsaI       pP6

Expected Outcome

The product of the reaction is a circular plasmid with a unique randomized promoter upstream of an amilGFP reporter.

Duriong transformation, typically only 1 plasmid molecule enters a cell, confirs resistance, and results in a colony on your petri dish. Thus, each colony on your transformation plate represents a unique promoter. When exposed to blue light, colonies fluoresce with varying intensity depending on promoter strength.

Figure: A pP6 transformation plate under blue light, showing colonies that express varying levels of GFP due to differences in promoter strength. The highlighted region is zoomed in to reveal diverse green intensities among clones, even though all share the core consensus motif. This illustrates the wide range of activities produced by flanking sequence variation—J23119 is unusually strong for this pattern.

Picking Colonies and Sequencing

You’ll select the brightest colonies (most green) for follow-up:

1. Grow in liquid culture
2. Miniprep DNA
3. Submit for sequencing
4. Analyze the sequence to identify the promoter

Note: Smaller, slow-growing colonies might encode the strongest promoters. Don’t skip them!

Getting Started

1. Download the pP6 lab sheets and sequences:
   Google Drive - pP6 Materials

2. Make a copy of this spreadsheet:
   pP6 LabSheet Workbook

3. Fill in your name and ID, and print the lab worksheets. You may also use your phone, tablet, or notebook. You may also write out your own versikon.

4. Read the tutorial and Watch the demo videos before each lab activity.

All Demo Videos

** replace with new vids as links - pP6-2022-1-PCR
- pP6-2022-2-Gel
- pP6-2022-3-Zymo
- pP6-2022-4-Assembly
- pP6-2022-5-Transformation
- pP6-2022-6-Pick
- pP6-2022-7-Miniprep
- pP6-2022-8-Sequencing
- pP6-2022-9-Sequence Analysis

Next

When you're ready, proceed to the Pipetting tutorial to learn how to use a micropipette correctly before starting wetlab work.

🧪 Quiz: Promoter Engineering Concepts

1️⃣ Fixed Motifs

Which promoter elements are fixed in each synthetic promoter?

Only the +1 site
The entire promoter
-35, -10, and +1
Just the -10 box

2️⃣ Randomized Regions

What part of the promoter is randomized?

The entire gene
The ribosome binding site
The flanking regions around -35, -10, +1
The coding sequence

3️⃣ Why Not J23100?

Why are J23100-family promoters avoided in pP6 for multi-gene constructs?

They are too weak
They don't work in E. coli
Their sequences are too similar, causing recombination
They lack RBS elements

Check Answers