Add to List

In this lab students get hands-on experience with cloning ribosomal binding sites onto a plasmid, then observe its effects on transformed bacteria compared to a control by quantifying the expression of GFP in red and/or blue colonies. Next, they use a thermocycler and the Golden Gate Assembly (GGA) technique to clone 2 different ribosome binding sites into a plasmid with a reporter gene. Finally, they transform their GGA reactions into E. coli. For 8 groups of 2 to 4 students.

Product Details
Advanced—For experienced high school and college classes; requires some technical skill.

Bring your biotechnology classroom instruction to the next level with a unique synthetic biology lab that focuses on the regulation of translation. Students get hands-on experience with molecular cloning when they add ribosomal binding sites (RBSs) to a plasmid and observe their effects on the expression of either a reporter gene that causes colonies to turn blue or one that causes them to glow red under UV or blue light. An additional reporter gene that causes green fluorescence protein expression is removed during the cloning process, providing students with another way to differentiate colonies containing original unaltered plasmid from those containing plasmids that have been changed during the closing process. This one-of-a-kind, colorful lab experience enables students to easily measure the effects of RBS sequences on bacterial protein production as they learn several of the core methods of recombinant DNA technology.

In this lab, students also use a method of recombinant DNA production called Golden Gate Assembly (GGA) to clone a weak and a strong RBS into either of 2 reporter gene expression units in a plasmid. They transform their GGA reactions into E. coli and observe the ability of each cloned RBS to support expression of the reporter gene. Students quantify reporter gene expression using photographs of their colonies on petri dishes with free, easy-to-use ImageJ software.

Product Features

• Designed for 8 groups of 2 to 4 students
• Teacher guide includes paper model activity to help visualize molecular processes
• Includes voucher to request perishable materials at a later date
• Comes with FREE 1-year access to digital resources that support 3-dimensional instruction for NGSS

Note: Kit includes a voucher to request perishable materials later at your convenience. Contact Carolina or return the voucher to request delivery of perishables.

Time Requirement
Lab, minimum of five 50-minute class periods (250 minutes total). Additional 110 minutes to produce representations of data and discuss outcomes at conclusion of lab.

Digital Resources
Includes 1-year access to digital resources that support 3-dimensional instruction for NGSS. Digital resources may include a teacher's manual and student guide, pre-lab activities and setup videos, phenomenon videos, simulations, and post-lab analysis and assessments.

Connection to the Next Generation Science Standards*
Science and Engineering Practices

• Analyzing and Interpreting Data
• Engaging in Argument from Evidence

Performance Expectation(s)

• HS-LS1-1: From Molecules to Organisms: Structures and Processes

Disciplinary Core Ideas

• LS1.A: Structure and Function
• LS3.A: Inheritance of Traits

Crosscutting Concepts

• Cause and Effect
• Scale, Proportion, and Quantity

Learning Objectives
Students:

• Manipulate gene regulation and can describe the role of ribosome binding sites in the initiation of translation in bacterial cells.
• Learn how cloning ribosome binding sites into a plasmid with reporter genes enables the measurement of RBS efficiencies, or strengths of function.
• Can explain how the cloning technique Golden Gate Assembly works.
• Are able to explain how the differences between type II and type IIS restriction enzymes contribute to Golden Gate Assembly cloning.
• Perform transformation of E. coli and use of selective media as fundamental techniques in molecular cloning.
• Can predict how well different cloned RBSs support translation by understanding the relationship between RBS sequence and ribosome function.
• Support claims about RBS strengths by measuring the levels of reporter proteins in E. coli colonies.

Prerequisite Knowledge and Skills
Students should possess a firm understanding of basic DNA structure, a solid understanding of genes and their function, and a good understanding of the processes by which genes are transcribed into RNA, which is translated to form proteins. Experience using a micropipette is useful.

*Next Generation Science Standards® is a registered trademark of WestEd. Neither WestEd nor the lead states and partners that developed the Next Generation Science Standards were involved in the production of this product, and do not endorse it.